Treatment of autoimmune diseases

ABSTRACT

Compositions can be used to stimulate growth of a hair shaft from a hair follicle. These compositions can include methylated polynucleotides useful in treatment of autoimmune diseases or conditions, including those, such as alopecia areata, that result in hair loss.

RELATED APPLICATION

This application claims the priority benefit of U.S. Provisional PatentApplication No. 61/800,354 filed on Mar. 15, 2013; and is acontinuation-in-part of PCT/US2012/056761, filed Sep. 21, 2012, whichclaims priority to U.S. Provisional Patent Application No. 61/538,682,filed Sep. 23, 2011; the entirety of each of these applications isincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 11, 2014, isnamed 088025-0077_ SL.txt and is 5,614 bytes in size.

FIELD

The subject technology relates generally to compositions and methods forthe treatment of autoimmune diseases or conditions, in particularautoimmune diseases or conditions that result in hair loss, such asalopecia.

BACKGROUND

Alopecia is a condition in which hair is lost. The loss of hair inalopecia is not limited just to head hair but can happen anywhere on thebody. Although not usually life-threatening, since it is accompanied byserious emotional distress due to issues related to appearance, there isa desire for an excellent agent for the treatment and an excellent agentfor the prevention of alopecia. Furthermore, since alopecia is oftenaccompanied by fading of hair color, there is a desire for an agent forthe prevention and an agent for the treatment of fading of hair coloraccompanying alopecia. Moreover, since alopecia is often accompanied bydeterioration of hair quality such as hair becoming finer or hairbecoming shorter, there is a desire for an agent for the prevention andan agent for the treatment of deterioration of hair quality accompanyingalopecia.

With regard to types of alopecia, there are alopecia areata,androgenetic alopecia, postmenopausal alopecia, female pattern alopecia,seborrheic alopecia, alopecia pityroides, senile alopecia, cancerchemotherapy drug-induced alopecia, alopecia due to radiation exposure,trichotillomania, postpartum alopecia, etc.

These types of alopecia have the same symptoms of hair loss, but arebased on different causes, and therapies therefor are different fromeach other. In particular, androgenetic alopecia, which is based on theaction of male hormone, and alopecia areata, which is suspected to be animmune disease, are very different diseases. Furthermore, it is thoughtthat postpartum alopecia, female pattern alopecia, seborrheic alopecia,alopecia pityroides, senile alopecia, cancer chemotherapy drug-inducedalopecia, and alopecia due to radiation exposure have different causesfrom each other, and there are hardly any effective therapies.

Alopecia areata is alopecia in which coin-sized circular to patchy baldarea(s) with a clear outline suddenly occur, without any subjectivesymptoms or prodromal symptoms, etc. in many cases, and subsequentlywhen spontaneous recovery does not occur they gradually increase in areaand become intractable. Alopecia areata is suspected to be an autoimmunedisease, but the cause thereof has not yet been discovered, and there isno known definite treatment method.

Alopecia areata is known to be associated with an autoimmune diseasesuch as a thyroid disease represented by Hashimoto's disease, vitiligo,systemic lupus erythematosus, rheumatoid arthritis, or myasthenia gravisor an atopic disease such as bronchial asthma, atopic dermatitis, orallergic rhinitis.

Androgenetic alopecia (AGA) is alopecia in which male hormone acts onmale hormone-sensitive hair follicles to form vellus hair, and occurs inabout half of males and 10% to 20% of females. It is thought thatgenetic predisposition is a large factor in androgenetic alopecia; inandrogenetic alopecia in males, the head hair on the frontal region andcrown becomes fine and short and turns into vellus hair, the hair lineon the forehead finally retreats, and head hair on the crown is lost. Onthe other hand, in androgenetic alopecia in females, in general thehairline does not change but hair of the entire head, in particular thecrown and the frontal region, becomes fine. Finasteride improves onlyabout ¼ of patients for androgenetic alopecia in males, and sinceadministration of finasteride to females is contraindicated, finasteridecannot be used for androgenetic alopecia in females.

Postpartum alopecia is alopecia in which hair whose growth phase hasbeen maintained by estrogen enters the resting phase all at once due tochildbirth, and hair loss increases. The hair loss of postpartumalopecia usually starts approximately 2 months after childbirth andcontinues until about 6 months after childbirth; since it usuallyrecovers within 1 year unless there is late childbearing, in most casesa treatment is not particularly required, but there are cases in whichhair does not recover spontaneously.

Female pattern alopecia is alopecia that is thought to occur due to adecrease in the amount of the female hormone estrogen relative to theamount of androgen in the bloodstream. It often occurs after themenopause, and in this case it is also called postmenopausal alopecia.Female pattern alopecia might be improved by hormone replacement therapybut is intractable in many cases.

Seborrheic alopecia is alopecia that is caused by excessive sebumsecretion on the scalp, pores are blocked thereby causing inflammationaround the pores or in the hair root, and the hair falls out. Seborrheicalopecia is improved to some extent by removing sebum by washing thehair, but it easily reoccurs and exhibits intractability.

Alopecia pityroides is alopecia that is caused by dandruff blockingpores to thus cause inflammation. Alopecia pityroides is often caused byexcessive hair washing; remission is achieved by reducing the number oftimes of hair washing or using a shampoo having a weak washing power,but it easily reoccurs and is intractable.

Trichotillomania is alopecia due to a hair-pulling disorder.Trichotillomania is a symptom resulting from pathological anxiety andcan be treated by behavioral therapy or psychological therapy.

Senile alopecia is alopecia in which, due to aging regardless of gender,body hair of the entire body, including all of the head hair, graduallybecomes thinner. It is thought that this is a natural phenomenon thatappears in many people due to aging, and this is not particularly atarget for treatment at the present. However, there is a desire forimprovement since there is an increased social requirement for improvingthe quality of life of elderly people accompanying a rise in the averagelifespan.

Cancer chemotherapy drug-induced alopecia is alopecia that is a sideeffect of anticancer treatment with a cancer chemotherapy drug. Theshock to the patient caused by loss of hair in all areas including notonly the head but also the eyebrows, eyelashes, nasal hair, underarmhair, and pubic hair is profound even if it is explained in advance.Since this also hinders the carrying out of cancer chemotherapy, thereis a high need for a treatment for this. Similarly, alopeciaaccompanying radiation exposure is also alopecia that occurs based onthe same mechanism as that for cancer chemotherapy drug-induced alopeciain terms of cancer cells being selectively killed by inhibiting celldivision. Therefore, a drug that can treat alopecia accompanying cancerchemotherapy can also treat alopecia accompanying radiation exposure.

In addition, alopecia due to an adverse reaction to a drug such as anantithyroid drug, an anticoagulant, thallium, a psychotropic drug, or aβ-blocker, alopecia due to a fungus, alopecia due to an endocrinedisorder such as dyspituitarism, hypothyroidism, or hyperthyroidism,alopecia due to a metabolic disorder such as a nutritional disorder,hypoalbuminemia, cachexia, iron-deficiency anemia, zinc deficiency,homocystinuria, or cirrhosis of the liver, toxic alopecia, alopecia dueto high temperature, childbirth, major surgery, sudden body weight loss,or serious illness, etc. are known, and they can be tackled by removingthe respective causes thereof.

Among these types of alopecia, female pattern alopecia, seborrheicalopecia, and alopecia pityroides can be tackled to some extent byremoving the respective causes thereof, but they easily reoccur, and areintractable. Furthermore, although it is thought that the cause offemale pattern alopecia is related to hormone balance, hormonereplacement therapy is indicated for menopausal disorders, osteoporosis,and hyperlipidemia but is not indicated for female pattern alopecia;since there is a possibility of cancer being caused by hormonereplacement therapy, hormone replacement therapy is not carried out forthe purpose of treating female pattern alopecia. Furthermore, withregard to androgenetic alopecia, there is no adequate therapy yet, andwith regard to alopecia areata, even the cause thereof is littleunderstood.

As described above, among the types of alopecia, the types of alopeciathat are difficult to treat are alopecia areata and androgeneticalopecia, and for alopecia areata in particular there are hardly anyeffective treatment methods. Moreover, there are hardly any therapiesfor postpartum alopecia, female pattern alopecia, alopecia pityroides,senile alopecia, and cancer chemotherapy drug-induced alopecia.

There is a need to develop therapeutic agents to treat hair loss.

SUMMARY

The subject technology generally relates to compositions and methods totreat hair loss or to stimulate the growth of hair.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of chromosomal integration of thesssI gene into E. coli.

FIG. 2 shows the treatment efficacy of type 1 diabetes using plasmid DNAmethylated with a DH5-alpha E. coli strain carrying a single copy of thesssI gene in its chromosome and the effect of methylation levels of theplasmid DNA vaccine on treatment efficacy. The graph illustrates theonset of diabetes in 16-week-old NOD mice receiving two different ratiosof hypo- or hyper-methylated, bivalent pro-apoptotic DNA vaccines. Mice(N=10/group) received a single weekly i.d. injection of the indicatedDNA at 16 weeks of age for 8 weeks and were monitored for non-fastingblood glucose >300 mg/dL. M: indicates methylated plasmid DNA; 4:1: 40and 10 μg, respectively, of the indicated plasmid DNA constructs; 4:2,40 and 20 μg, respectively, of the indicated plasmid DNA constructs; *:P<0.05 (Kaplan-Meier).

FIG. 3 shows the effects of DNA methylation levels on CD8+ T cellsinfiltration in pancreatic islets of immunized female non-obese diabetic(NOD) mice (Type 1 diabetes (T1D) model). Plasmid DNA was methylatedusing a DH5-alpha E. coli strain carrying a single copy of the sssI genein its chromosome. FIG. 3 shows that increased DNA methylation causesdecreased infiltration of islets by CD8*+ T lymphocytes. CpG-methylationcauses an antigen-dependent increase in percentage of CD4+CD25+Foxp3+ invitro. *, P<0.05.

FIG. 4 shows the effects of DNA methylation levels in percent of cellswith Tregulatory cell phenotype in lymph nodes of immunized female NODmice (T1D model). Plasmid DNA was methylated with a DH5-alpha E. colistrain carrying a single copy of the sssI gene in its chromosome. Thedata in FIG. 4 show that increased DNA vaccine methylation causes anincreased percent of cells with Treg phenotype. *, P<0.05,

FIG. 5 shows the effect of CpG methylation of plasmid DNA on skinallograft survival. Plasmid DNA was methylated with a DH5-alpha E. colistrain carrying a single copy of the sssI gene in its chromosome. *,P<0.05, Kaplan Meyer. *, P<0.05,

FIG. 6 illustrates the effect of DNA methylation on skin allografts.Only the methylated BAX DNA results in hair growth after 5 weeks.

FIG. 7 shows that plasmid DNA hypermethylation causes increasedrecruitment of total DCs andpDCs. NOD mice (N=15/group) received i.d.injection of hypomethylated vector, hypomethylated (sGAD+BAX), partiallyhypermethylated (msGAD+BAX), or fully hypermethylated (msGAD+mBAX)plasmid DNA. Total DCs were isolated from pooled LNs using differentialcentrifugation, and counted (70-80% were CD11c+ as per flow cytometricanalysis). A portion of total DCs was then used to isolate and countmPDCA-1+pDCs using a MACs kit (Miltenyi Biotec, Auburn, Calif.). *,P<0.02 vs vector, **, P<0.008 vs msGAD+mBAX, ***, P<0.002 vs sGAD+BAX.

FIG. 8 shows that plasmid DNA hypermethylation affects Treg populationand activity. CD4+CD25+ and CD4+CD25− T lymphocytes were isolated fromspleen of NOD mice (N=6-8/group) immunized with the indicated DNAconstructs (2:1 ratio) with increased levels of CpG methylation (m).Cells were then co-transferred i.v. with diabetogenic NOD T lymphocytesinto NOD-scid mice (N=6/group) to investigate diabetes suppression andTreg activity. Contr, cells isolated from untreated mice, Vac, cellsisolated from mice receiving the indicated DNA. *, P<0.05, Kaplan-Meier.

FIG. 9 shows that SKRS95 causes decreased percentage of both Th17 andTc17 cells in cultured islets. Hand-picked islets were isolated from NODmice receiving the indicated DNA (N=6/group, ˜100 islets/mouse). Isletswere cultured with IL-2 for 7 days, dispersed, and stained for CD4, CD8and intracellular IL-17 with immunofuorescent mAbs for flow cytometricanalysis. *, P<0.05.

FIG. 10 shows that co-injection of CpG oligonucleotide causes a decreasein suppressive activity of pDCs induced by mSGAD+BAX DNA. 7-week-oldfemale NOD mice (N=15/group), received 60 micrograms of the indicatedDNA intradermally, i.e., hypomethylated sGAD+BAX DNA (2:1) or partiallyhypermethylated msGAD+BAX DNA (2:1) and 20 micrograms of oligonucleotideinhibiting binding of unmethylated CpG DNA to TLR9 (G), or 20 microgramsof CpG oligonucleotide binding to TLR9 (CpG) 3 times over 2 weeks.Axillary and pancreatic draining LN were pooled from 15 mice/group, andspleens were pooled from 4 untreated mice. LN DCs were pre-enrichedusing density centrifugations and LN pDCs were isolated using a pDC kit(Miltenyi, Auburn, Calif.). Untreated spleen Pan T cells were isolatedusing a Pan-T negative kit and stained with 1.5 μM CFSE. For each setcells were loaded as 100 ul DC+100 ul TC/well in 96-well-plates fortotally 3 wells/Ag, 3 wells for LPS, 3 wells for Ins, and the last 3wells for GAD antigen stimulation. Cells were cultured for 3 days withhrIL2. Cells were then collected and FACS was performed forCFSE+CD4+SYTOX− cells. pDC or cDC from vaccinated NOD and CFSE labeled(1.5 uM) Pan T cells from untreated NOD were added as 1:1 onto 96-wellplate with IL-2 20 U/ml, and LPS 5 ug/ml, or Ins 20 ug/ml, or GAD 20ug/ml as antigen stimulation, or without antigen. Cells were cultured incomplete medium in a CO2 incubator for 3 days, and anti-CD4-PE Abs andSytox were used to detect CFSE+CD4+Sytox− cell proliferations. FlowJo7.6.5 software was used to analyze the proliferation data, and % Dividedwas used to represent differences between groups

DETAILED DESCRIPTION

1. Overview

The subject technology generally relates to compositions and methods totreat hair loss or to stimulate the growth of hair.

As described and exemplified herein, hair loss, such as alopecia areata,is a condition often attributable to an autoimmune disease that targetshair follicles. By delivering a pro-apoptotic protein (e.g., to the siteof inflammation or the site of hair loss), tolerogenic apoptosis of hairfollicles is induced by the pro-apoptotic protein. As a result, theapoptotic follicles containing autoantigen(s) are produced, which inturn induce a regulatory response specific for hair follicles. Theregulatory response induces immune tolerance to autoantigen(s), therebyreducing the abnormal autoimmune response that attacks hair folliclecells.

Bacterial DNA contains low levels of methylated CpG dinucleotidescompared to mammalian DNA. The mammalian immune system uses this featureof bacterial DNA as a signal to identify foreign DNA and respond tothreats by bacterial pathogens. Accordingly, unmethylated CpGdinucleotides can serve as adjuvant for DNA vaccines engineered toinduce effector responses against pathogens. In contrast, unmethylatedCpG dinucleotides are detrimental for plasmid DNA vaccines engineered toinduce an immunoregulatory response to suppress and control inflammationin disorders like autoimmune disease, allergy, asthma, organ transplantrejection, and cancer. Moreover, presence of unmethylated CpGdinucleotides in plasmid DNA also shortens time of gene expression inanimals, which can be detrimental to gene therapy approaches.Accordingly, methylation of CpG dinucleotides could be used to enhancethe potency of DNA vaccines for immune-mediated inflammatory disorders,to increase the time of therapeutic gene expression for gene therapy,and to enable multiple dosing without an immune-based adverse reaction.

Currently, it is possible to generate CpG-methylated plasmid DNA afteramplification in bacterial strains carrying plasmid DNA encoding anenzyme like the SssI methylase, or to use the purified enzymes tomethylate plasmid DNA in vitro. However, in vitro plasmid methylationcannot be envisaged for the industrial production of a plasmid whichwould be used in gene therapy. A method of plasmid DNA production must,in effect, enable large and homogeneous amounts of plasmids to beproduced reproducibly, cost effectively, and this DNA to be purified bymethods which are acceptable for pharmaceutical application.

Similarly, amplification of hypermethylated plasmid DNA in bacteriausing a plasmid DNA construct encoding the methylase of interest cannotbe readily translated to human therapeutics because of contaminationwith the methylase-encoding plasmid DNA, and the possibility ofinconsistent methylation of the DNA produced resulting from variation inthe copy number of the methylase-encoding plasmid DNA. The use of aplasmid DNA or expression cassette containing a gene encoding methylaseM. SssI in bacterial cells under the control of an IPTG induciblepromoter, where greater than 90% of the cytosines are methylated, iscurrently possible.

The incorporation of the SssI methylase gene into the E. coli chromosomeunder the control of an inducible promoter (arabinose-induciblepromoter, PBAD), where greater than 90% of CpG sites are methylated, isalso currently possible. However, over-methylation can lead toproduction of plasmid DNA that is not optimal for gene therapy or immunestimulation, as will be shown in the present disclosure. Accordingly,above described inducible system is inappropriate to make consistentplasmid products with intermediate methylation, e.g., 30-50%. Moreover,the inducible system introduces additional variables that createuncertainty in the reproducible production of methylated DNA plasmid DNAfor commercial gene therapy applications. For example, the timing ofinduction for a specific batch could significantly impact levels ofplasmid DNA methylation.

Alternatively, CpG dinucleotides present in plasmid DNA can be replacedwith other nucleotides for human application but this requirestime-consuming genetic engineering and trial and error testing. Inaddition, the promoter sequence and the sequence of the gene of interestmay not function optimally after genetic engineering to remove the CpGdinucleotides. Thus, the replacement of CpG methodology is not readilyapplicable to any plasmid DNA construct of choice. Lastly, for humangene therapy applications, the modified promoter sequence and modifiedgene of interest raise additional issues and uncertainty with regard tosafety and efficacy.

The construction of E. coli strains carrying chromosomal copies of aCpG-methylase gene under control of a constitutive promoter circumventsthese problems and is described in detail herein.

Further, different levels of DNA methylation (e.g., about 50% CpGmethylation, about 40% CpG methylation, about 30% CpG methylation, about25% CpG methylation, etc.) may be desired for different applications.Levels of methylation can be adjusted by multiple methods, such as bymodulating the expression level of methylase in the bacterial host (forexample, by using promoters of different strength).

In another aspect, as described and exemplified herein, the inventorsdiscovered that superior results were achieved by mixing hypomethylatedplasmid DNA (e.g., around 10-15% CpG methylation, or lower) encodingpro-apoptotic protein (BAX), and hypermethylated plasmid DNA (e.g., 50%CpG methylation) encoding an antigen, to induce immune tolerance to saidantigen. The antigen may be encoded by a plasmid DNA as exemplifiedherein, or it may be co-injected with a plasmid DNA, or present at thesite of injection of a plasmid DNA, provided that it is present orsynthesized in sufficient amounts. Data presented herein demonstratethat a mixture of hypomethylated and hypermethylated plasmid DNAsachieved higher efficacy, as compared to hypomethylated orhypermethylated plasmid DNA alone, in treating type 1 diabetes (T1D) innon-obese diabetic (NOD) mice. Accordingly, hypomethylated andhypermethylated DNA may be mixed to achieve a desired methylation levelfor a specific therapeutic application.

In another aspect, the subject technology provides an isolated bacteriumwith chromosomal DNA comprising a toxic engineered gene controlled by aconstitutive promoter stably incorporated into the chromosomal DNA. Invarious embodiments, the bacterium comprises E. coli. In variousembodiments, the toxic engineered gene of the E. coli bacteria describedherein comprises a DNaseI gene or a gene encoding an HIV-1 protease.

As described and exemplified herein, the inventors discovered that whenthe methylase gene sssI was introduced into an E. coli cell in the formof a plasmid, such that the plasmid was replicated in vivo, themethylase gene became toxic to the bacterial host. It is believed thatwhen the plasmid was replicated in vivo, methylase was expressed at adose that caused toxicity, killing the host cells before the plasmidcould be integrated into the genome. Accordingly, the inventorsreplicated the plasmid in vitro, such that enough copies of sssI geneswere directly introduced into the host cell without the need for in vivoreplication. By this way, the sssI gene was integrated into the genomewithout the requirement of in vivo replication. Because of the low copynumber of genome-integrated sssI gene (as compared to a copy numberplasmid), the amount of methylase produced this way did not reach thetoxic level. This method can be used to recombinantly express othergenes that are toxic to the host.

Accordingly, some methods of the subject technology further includeincorporating a gene toxic to an Escherichia coli (E. coli) bacteriacontrolled by a constitutive promoter, the method comprising (a)selecting a plasmid containing the gene toxic to E. coli and aselectable marker in the proper orientation; (b) amplifying the plasmidin vitro to produce a microgram quantity of the plasmid; (c)electroporating the plasmid into the E. coli; and (d) selecting the E.coli incorporating the gene toxic to the E. coli. Some methods providethat the amplifying is performed by a rolling circle amplification. Somemethods provide that the gene toxic to E. coli comprises a methylasegene.

While the subject technology is capable of being embodied in variousforms, the description below of several embodiments is made with theunderstanding that the present disclosure is to be considered as anexemplification of the subject technology, and is not intended to limitthe subject technology to the specific embodiments illustrated. Headingsare provided for convenience only and are not to be construed to limitthe subject technology in any manner. Embodiments illustrated under anyheading may be combined with embodiments illustrated under any otherheading.

2. Definitions

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” Slight variationsabove and below the stated ranges may be used to achieve substantiallythe same results as values within the ranges. The disclosure of rangesis intended as a continuous range including every value between theminimum and maximum values recited as well as any ranges that can beformed by such values. Accordingly, the skilled person will appreciatethat many such ratios, ranges, and ranges of ratios can be unambiguouslyderived from the data and numbers presented herein and all representvarious embodiments of the subject technology.

As used in this disclosure, the term “autoantigen” means and includes anendogenous antigen that stimulates the production of autoantibodies, asin an autoimmune reaction, as well as part of such endogenous antigens,or modified endogenous antigens that elicit the same response as thefull endogenous antigen, as will be understood by those with skill inthe art with reference to this disclosure. For example, in the contextof this disclosure carbonic anhydrase II, chromogranin, collagen, CYP2D6(cytochrome P450, family 2, subfamily Device 400, polypeptide 6),glutamic acid decarboxylase, secreted glutamic acid decarboxylase 55,hCDR1, HSP60, IA2, IGRP, insulin, myelin basic protein, hNinein, Ro 60kDa, SOX-10 (SRY-box containing gene 10), ZnT8, and the like, areautoantigens. Also encompassed are antigenic fragments of the any one ofthe foregoing autoantigens.

The term “about”, as used here, refers to +/−5% of a value.

The term “functional fragment” of protein refers to a peptide fragmentthat is a portion of the full length protein, and has substantially thesame biological activity, or carries out substantially the same functionas the full length protein (e.g., carrying out the same enzymaticreaction). For example, a functional fragment of a pro-apoptotic proteincan promote the apoptosis of a cell.

The term “hypermethylated” (sometimes abbreviated as “methylated”) whenused in reference to a DNA, means that at least about 30% (preferably atleast about 35%, or at least about 40%, or at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%) of the CpG dinucleotides are methylated. Forexample, mammalian DNA, in which between 60% and 90% of all CpGs aremethylated, is hypermethylated.

The term “hypomethylated” (sometimes abbreviated as “unmethylated”) whenused in reference to a DNA, means that about 15% or less (preferablyabout 10% or less, about 7.5% or less, about 5% or less, or about 3%, orabout 1% or less) of the CpG dinucleotides are methylated. For example,bacterial DNA, in which between 5% and 15% of all CpGs are methylated,is hypomethylated.

A “methylation” level, when used in reference to a DNA, refers to thepercentage of methylated CpG dinucleotides out of the total CpGdinucleotides of the DNA molecule.

The term “controlled” means “operably linked,” which refers to theassociation of a nucleic acid sequence on another nucleic acid fragmentso that the function of one is affected by the other. For example, acoding sequence is “controlled” by a promoter when the promoter iscapable of affecting the expression of that coding sequence (i.e., thatthe coding sequence is under the transcriptional control of thepromoter). Coding sequences can be operably linked to regulatorysequences in sense or antisense orientation.

As used in this disclosure, the term “comprise” and variations of theterm, such as “comprising” and “comprises,” are not intended to excludeother additives, components, integers, or steps.

As used in this disclosure, the term “constitutive promoter” means andincludes promoters that express a gene whether or not an inducer ispresent.

As used in this disclosure, the term “donor antigen” means and includesan antigen from an allograft that was transplanted into the organism totake the place of defective or absent cells or tissues, such as forexample, islet cell transplants, and partial or whole organ transplantsincluding transplanted hearts, lungs, kidneys and livers, and thatstimulates the production of antibodies and leukocytes that produce animmune reaction, as well as part of such donor antigens, or modifieddonor antigens that elicit the same response as the full donor antigen,as will be understood by those with skill in the art with reference tothis disclosure. Also encompassed are antigenic fragments of the any oneof the foregoing donor antigens.

As used in this disclosure, the term “immune-mediated inflammatorydisorders” means and includes both diseases due in part or in total todestruction of normal cells or tissues by the immune system of theorganism, and also comprises destruction by the immune system of theorganism of cells or tissues (allografts) that were transplanted intothe organism to take the place of defective or absent cells or tissues,such as for example, islet cell transplants, and partial or whole organtransplants including transplanted hearts, lungs, kidneys and livers.The immune-mediated inflammatory disorder may be, for example, therejection of solid organ transplants, graft versus host disease, hostversus graft disease, autoimmune hepatitis, vitiligo, diabetes mellitustype 1, Addison's Disease, Graves' disease, Hashimoto's thyroiditis,multiple sclerosis, polymyalgia rheumatica, Reiter's syndrome, Crohn'sdisease, Goodpasture's syndrome, Gullain-Barré syndrome, lupusnephritis, rheumatoid arthritis, systemic lupus erythematosus, Wegener'sgranulomatosis, celiac disease, dermatomyositis, eosinophilic fasciitis,idiopathic thrombocytopenic purpura, Miller-Fisher syndrome, myastheniagravis, pemphigus vulgaris, pernicious anaemia, polymyositis, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis,Sjögren's syndrome, and the like.

As used in this disclosure, the term “DNA vaccine” means and includesDNA sequences that code for immunogenic proteins located inappropriately constructed plasmids, which include strong promoters,which when injected into an animal are taken up by cells and theimmunogenic proteins are expressed and elicit an immune response.

As used in this disclosure, the term “gene therapy” means and includescorrecting or ameliorating a deficiency or an abnormality by introducinggenetic information into either an affected cell or organ, or into anon-affected cell or organ, so as to correct or ameliorate thedeficiency and abnormality as a result of the gene therapy. Thisinformation may be introduced either in vitro into cells extracted fromthe organ or other cells such as a stem cell, and then re-injecting themodified into the body, ex vivo by introduction to a removed tissue ororgan; or in vivo, directly into the target tissue.

3. Bacterial Strains Expressing Methylase

In one aspect, the subject technology provides an isolated bacteriumcomprising an engineered polynucleotide sequence encoding a methylasecontrolled by a constitutive promoter, wherein said engineeredpolynucleotide is stably incorporated into the chromosomal DNA of saidbacterium.

Suitable bacteria include, e.g., Bacillus brevis, Bacillus megaterium,Bacillus subtilis, Caulobacter crescentus, other strains, or,Escherichia coli. In certain embodiments, the bacterium is E. coli.

Methylase, also known as methyltransferase (MTase), is a type oftransferase enzyme that transfers a methyl group from a donor to anacceptor. All the known DNA methyltransferases use S-adenosyl methionine(SAM) as the methyl donor.

MTases can be divided into three different groups on the basis of thechemical reactions they catalyze: 6A—those that generateN6-methyladenine (EC 2.1.1.72); m4C—those that generateN4-methylcytosine (EC 2.1.1.113); and m5C—those that generateC5-methylcytosine (EC 2.1.1.37). m6A and m4C methyltransferases arefound primarily in prokaryotes. m5C methyltransfereases are found insome lower eukaryotes, in most higher plants, and in animals beginningwith the echinoderms.

Nucleic acid sequences encoding various MTase have been found in manypublished genome sequences. See e.g., REBASE database(http://rebase.neb.com/rebase); or GenBank database(http://www.ncbi.nlm.nih.gov/genbank).

In one embodiment, the subject technology provides an isolated bacteriumwith chromosomal DNA comprising an engineered methylase gene controlledby a constitutive promoter stably incorporated into the chromosomal DNA.In various embodiments, the bacterium comprises E. coli. In variousembodiments, the methylase gene of the E. coli bacteria described hereincomprises a CpG methylase gene, M. CviPI gene, an M. AluI gene, an M.BamHI gene, an M.dam gene, a DnmtI gene, an M. EcoRI gene, an M. HaeIIIgene, an M. HhaI, an M.HpaII gene, an M. MspI gene, an M. TaqI gene, anM.G9a gene, an M. PRMT1 gene, or an M. SET7 gene. In some embodiments,the CpG methylase gene comprises an SssI gene. A methylase geneencompasses genomic form, cDNA form, or variants of genomic form or cDNAform (e.g., mutants or fragments) of a nucleic acid that encodes amethylase.

B. Expression Level of Methylase

As described herein, different levels of DNA methylation (e.g., about50% CpG methylation, about 45% CpG methylation, about 40% CpGmethylation, about 35% CpG methylation, about 30% CpG methylation, about25% CpG methylation, about 20% CpG methylation, about 15% CpGmethylation, etc.) may be desired for different applications. In oneaspect, levels of methylation can be adjusted by modulating theexpression level of a methylase in the bacterial host. For example, ifhigh level of DNA methylation is desired, a strong promoter may be usedto increase the expression level of methylase.

Expression level of a methylase in the bacterial host can be modulatedseveral ways. The transcriptional promoter and terminator sequences, theribosome-binding site (RBS) and the efficiency of translation in thehost organism, the intrinsic stability of the protein within the cell,etc, can affect the expression level, and can be manipulated usingart-known methods. For example, transcriptional regulatory sequences,such as promoters, enhancers or other expression control elements, areknown in the art (see, e.g., Goeddel, Gene Expression Technology Methodsin Enzymology 185, Academic Press (1990)). It will be appreciated bythose skilled in the art that the design of the expression vector,including the selection of regulatory sequences may depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, etc.

In certain embodiments, the expression level of the methylase (hence themethylation level of a DNA) is modulated by the strength of thepromoter. Many promoter sequences suitable for bacterial hosts are knownin the art, such as T3, T5, T7, Lac, lacZ, Trp, Gpt, lambda, PR, PL. Thestrengths of bacterial promoters have been reported. See e.g., Eeuschleet al., Promoters of Escherichia coli: a hierarchy of in vivo strengthindicates alternate structures, The EMBO Journal, 5:2987-2994 (1986);Rhodius et al., Predicting strength and function for promoters of theEscherichia coli: alternative sigma factor, PNAS, 107: 2854-2859 (2010).Liang et al. (Activities of constitutive promoters in Escherichia coli.,J Mol Biol. 1999 Sep. 10; 292(1):19-37) discloses seven constitutivepromoters in Escherichia coli. The promoters include (i) the spcribosomal protein operon promotor Pspc; (ii) the beta-lactamase genepromotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda;(iv) and (v) the replication control promoters PRNAI and PRNAII ofplasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnBribosomal RNA operon. The strength of the promoters are also disclosed.All of these promoters are suitable for use for the subject technology.

Alternatively or in addition, the expression level of the methylase(hence the methylation level of a DNA) can modulated by theribosome-binding site (RBS). Generally, the stronger the binding of themRNA to the ribosomal RNA, the greater the efficiency of translationalinitiation. Activity of a RBS can be influenced by the length andnucleotide composition of the spacer separating the RBS and theinitiator AUG. Bacterial mRNAs that do not have a close match to theconsensus ribosome attachment sequence are not translated efficiently.

The expression of methylase in a microbial host described herein can befurther improved by codon-optimization. For example, modifying aless-common codon with a more common codon may affect the half-life ofthe mRNA or alter its structure by introducing a secondary structurethat interferes with translation of the message. All or a portion of acoding-region can be optimized. In some cases the desired modulation ofexpression is achieved by optimizing essentially the entire gene. Inother cases, the desired modulation will be achieved by optimizing partof but not entire sequence of the gene.

The half-life of methylase may also be modulated, for example, by createa fusion protein in which the methylase is fused with a stable hostprotein.

4. Incorporating Methylase-Coding Sequence into Host Chromosome

Methods of incorporating the methylase-coding sequence (together withany regulatory sequences (such as transcriptional promoters andterminators, RBS, etc.) if desired) are known in the art. See, e.g.,U.S. Pat. Nos. 5,695,976, 5,882,888. Generally, for integration, theexogeneous DNA sequence should have some sequence homology forrecombination between the exogeneous DNA and the hose genome; thechromosome integration site should not be within an essential codinggene.

For example, a segment of DNA from the host chromosome can be cloned ona plasmid. The methylase-coding sequence (together with any regulatorysequences (such as transcriptional promoters and terminators, RBS, etc.,if desired) can be inserted in the middle of this chromosome sequence.Homologous DNA pairing will then occur between plasmid-born hostsequence and the host chromosome. A double cross over event will resultin the integration of the methylase-coding sequence. In addition tosequences encoding the methylase and regulatory sequences, the plasmidmay carry additional sequences, such as sequences that regulatereplication of the vector in host cells (e.g., origins of replication)and selectable marker genes.

As described and exemplified herein, the inventors discovered that whenthe methylase gene sssI was introduced into an E. coli cell in the formof a plasmid, such that the plasmid was replicated in vivo, themethylase gene became toxic to the bacterial host. It is believed thatwhen the plasmid was replicated in vivo, methylase was expressed at adose that caused toxicity, killing the host cells before the plasmidcould be integrated into the genome. Accordingly, the inventorsreplicated the plasmid in vitro, such that enough copies of sssI geneswere directly introduced into the host cell without the need for in vivoreplication. By this way, the sssI gene was integrated into the genomewithout the requirement of in vivo replication. Because of the low copynumber of genome-integrated sssI gene (as compared to a copy numberplasmid), the amount of methylase produced this way did not reach thetoxic level.

Accordingly, some methods of the subject technology further includeincorporating a gene toxic to an Escherichia coli (E. coli) bacteriacontrolled by a constitutive promoter, the method comprising (a)selecting a plasmid containing the gene toxic to E. coli and aselectable marker in the proper orientation; (b) amplifying the plasmidin vitro to produce a microgram quantity of the plasmid; (c)electroporating the plasmid into the E. coli; and (d) selecting the E.coli incorporating the gene toxic to the E. coli. Some methods providethat the amplifying is performed by a rolling circle amplification. Somemethods provide that the gene toxic to E. coli comprises a methylasegene.

This method can be used to recombinantly express other genes that aretoxic to the host. The toxicity of a recombinantly expressed protein canbe influenced by, for example, the strength of the promoter, the copynumber of the plasmid, the expression level of the protein, and thefunction of the protein, etc. Toxicity of a protein, and the thresholdlevel of a toxic protein before it becomes detrimental to a host, can beassessed, for example, by determining the growth rate of the bacterialculture. As used herein, a gene is considered toxic or reaches a toxiclevel when the bacterial host, when expressing the gene beyond athreshold level, cannot divide or replicate to produce daughter cells.For example, a gene may be considered as a toxic gene if it cause thebacterial host to stop cell division when more than 1 copy, more than 2copies, more than 3 copies, more than 4 copies, more than 5 copies, morethan 10 copies, more than 15 copies, more than 20 copies, more than 30copies, more than 40 copies, or more than 50 copies, of the gene arepresent in the bacterial host.

Site-specific integration is preferred over random integration, as theenvironment (sequence) of the integration site is known. The chromosomeintegration site disclosed in the Example is an “att” site. See,Haldimann et al., Conditional-Replication, Integration, Excision, andRetrieval Plasmid-Host Systems for Gene Structure-Function Studies ofBacteria, Journal of Bacteriology, vol 183, 6384-6393 (2001). The attsite allows specific integration of the methylase coding sequence intothe E. coli genome in single copies.

The plasmid may be transformed or transfected into a host cell bystandard techniques, such as electroporation, calcium-phosphateprecipitation, or DEAE-dextran transfection.

Bacterial strains described herein may be cultured in a suitable culturemedium known in the art. Descriptions of culture media for variousmicroorganisms can be found in the textbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981; the entirety of which is herebyincorporated herein by reference). These media which can be employed inaccordance with the subject technology usually comprise one or morecarbon sources, nitrogen sources, inorganic salts, vitamins and/or traceelements.

5. Use of Bacterial Strains

In another aspect, the bacterial strains described herein can be used toproduce DNA with a desired methylation level. For example, a DNAmolecule, such as a plasmid, produced by the bacterial strains describedherein will have a methylation level that is closer to a mammalian DNA,thereby reducing the risk of triggering an immune response.

Some methods of the subject technology include preparing a modifiedplasmid for use as a DNA vaccine or a gene therapy agent comprisingexpressing a plasmid encoding a first gene in a bacterium havingchromosomal DNA that comprises an engineered methylase gene controlledby a constitutive promoter stably incorporated into the chromosomal DNA.Some methods provide that the bacterium is E. coli.

In some embodiments, from about 10% to about 90% of the CpGdinucleotides of the DNA (such as a plasmid) produced by the bacterialstrain described herein are methylated. For example, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, from about 10% toabout 90%, from about 15% to about 80%, from about 15% to about 70%,from about 15% to about 60%, from about 15% to about 50%, from about 10%to about 50%, from about 15% to about 45%, from about 10% to about 45%,from about 15% to about 40%, from about 10% to about 40%, from about 15%to about 35%, from about 10% to about 35%, from about 15% to about 30%,from about 10% to about 30%, from about 15% to about 25%, from about 10%to about 25%, from about 25% to about 50%, from about 25% to about 45%,from about 25% to about 40%, from about 25% to about 35%, or from about25% to about 30%, of the CpG dinucleotides of the DNA (such as aplasmid) produced by the bacterial strain described herein aremethylated.

DNA molecules, such as plasmids, produced by the bacterial strainsdescribed herein can also be used for treating allergy, an autoimmunedisease, or cancer. Some methods provide that the DNA molecule comprisesa gene encoding at least one of an allergen, an autoantigen, a cancerantigen, a donor antigen, or a pro-apoptotic protein. Some methodsprovide that the DNA molecule comprises a polynucleotide sequenceencoding an allergen or an antigenic fragment thereof, an autoantigen oran auto-antigenic fragment thereof, a cancer antigen or an antigenicfragment thereof, a donor antigen or an antigenic fragment thereof, or apro-apoptotic protein or a functional fragment thereof. Any combinationsof these proteins or fragments are also encompassed.

Autoantigens include any self antigens that the host or patient immunesystem recognizes and responds against as foreign including, e.g., selfantigens associated with an autoimmune disorder. Some methods providethat the autoantigen is selected from the group consisting of carbonicanhydrase II, chromogranin, collagen, CYP2D6 (cytochrome P450, family 2,subfamily Device 400, polypeptide 6), glutamic acid decarboxylase,secreted glutamic acid decarboxylase 55, hCDR1, HSP60, IA2, IGRP,insulin, myelin basic protein, hNinein, Ro 60 kDa, SOX-10 (SRY-boxcontaining gene 10), and ZnT8. An antigenic fragment (such as anepitope) of any one of these auto-antigens is also encompassed.Additional examples of autoantigens are listed below.

Some methods provide that the allergen is selected from the groupconsisting of peanut allergens Ara h 1, 2 and 3; pollen allergens Phl p1, 2, 5a, 5b, 6, and Bet v 1; and cat allergen Fel d 1. Some methodsprovide that the donor antigens are a major or a minorhistocompatibility complex molecule. An antigenic fragment of any one ofthese allergens is also encompassed.

Some methods provide that the cancer antigen is selected from the groupconsisting of HER-2, gp100, melan A and PSA. Other cancer or tumorantigens include, e.g., (a) cancer-testis antigens such as NY-ESO-1,SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, forexample, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6,and MAGE-12 (which can be used, for example, to address melanoma, lung,head and neck, NSCLC, breast, gastrointestinal, and bladder tumors), (b)mutated antigens, for example, p53 (associated with various solidtumors, e.g., colorectal, lung, head and neck cancer), p21/Ras(associated with, e.g., melanoma, pancreatic cancer and colorectalcancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with,e.g., melanoma), caspase-8 (associated with, e.g., head and neckcancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701,beta catenin (associated with, e.g., melanoma), TCR (associated with,e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g.,chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205,CDC-27, and LDLR-FUT, (c) over-expressed antigens, for example, Galectin4 (associated with, e.g., colorectal cancer), Galectin 9 (associatedwith, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g.,chronic myelogenous leukemia), WT 1 (associated with, e.g., variousleukemias), carbonic anhydrase (associated with, e.g., renal cancer),aldolase A (associated with, e.g., lung cancer), PRAME (associated with,e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lungand ovarian cancer), alpha-fetoprotein (associated with, e.g.,hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), p53(associated with, e.g., breast, colon cancer), and carcinoembryonicantigen (associated with, e.g., breast cancer, lung cancer, and cancersof the gastrointestinal tract such as colorectal cancer), (d) sharedantigens, for example, melanoma-melanocyte differentiation antigens suchas MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma), (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer, (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example), and (g)other tumor antigens, such as polypeptide- and saccharide-containingantigens including (i) glycoproteins such as sialyl Tn and sialyl Lewisx (associated with, e.g., breast and colorectal cancer) as well asvarious mucins; glycoproteins are coupled to a carrier protein (e.g.,MUC-1 are coupled to KLH); (ii) lipopolypeptides (e.g., MUC-1 linked toa lipid moiety); (iii) polysaccharides (e.g., Globo H synthetichexasaccharide), which are coupled to a carrier proteins (e.g., to KLH),(iv) gangliosides such as GM2, GM12, GD2, GD3 (associated with, e.g.,brain, lung cancer, melanoma), which also are coupled to carrierproteins (e.g., KLH). An antigenic fragment of any one of these cancerantigens is also encompassed.

Some methods provide that the pro-apoptotic protein is selected from thegroup consisting of BAK, BAX, BIM, a modified caspase, Death Receptor 3(DR3), Death Receptor 4 (DR4), Death Receptor 5 (DR5), a FAS receptor, amodified survivin protein, and a Tumor Necrosis Factor Receptor. Afunctional fragment of any one of these pro-apoptotic proteins is alsoencompassed.

Some methods described here provide ways for increasing the expressionof regulatory T cells (Tregs) in a mammal comprising contacting themammal with a modified plasmid generated in a bacterium havingchromosomal DNA comprising an engineered methylase gene controlled by aconstitutive promoter stably incorporated into the chromosomal DNA. Somemethods provide that the bacterium is E. coli.

Some methods provide that the modified plasmid encodes an allergen, anautoantigen, a donor antigen, or a pro-apoptotic protein. Some methodsprovide that the DNA molecule comprises a polynucleotide sequenceencoding an allergen or an antigenic fragment thereof, an autoantigen oran auto-antigenic fragment thereof, a cancer antigen or an antigenicfragment thereof, a donor antigen or an antigenic fragment thereof, or apro-apoptotic protein or a functional fragment thereof. Any combinationsof these proteins or fragments may also be used.

Some methods provide that the autoantigen is selected from the groupconsisting of carbonic anhydrase II, chromogranin, collagen, CYP2D6(cytochrome P450, family 2, subfamily Device 400, polypeptide 6),glutamic acid decarboxylase, secreted glutamic acid decarboxylase 55,hCDR1, hNinein, HSP60, IA2, IGRP, insulin, myelin basic protein, Ro 60kDa, SOX-10 (SRY-box containing gene 10), and ZnT8. Antigenic fragmentsof the foregoing autoantigens may also be used. Other autoantigensdescribed herein may also be used.

Some methods provide that the pro-apoptotic protein is selected from thegroup consisting of BAK, BAX, BIM, a modified caspase, Death Receptor 3(DR3), Death Receptor 4 (DR4), Death Receptor 5 (DR5), a FAS receptor,modified surviving protein, and Tumor Necrosis Factor Receptor. Afunctional fragment of any one of these pro-apoptotic proteins may alsobe used.

For different purposes, the methylation level may need to be adjusted oroptimized. For example, U.S. Pat. Pub. No. 2009/0191218 (Escher et al.)discloses the use of methylated plasmid for treating skin graft. In theskin graft model reported therein, about 50% of the CpG dinucleotides inthe plasmid were methylated. The plasmid was effective in treating skintransplant rejection. In comparison, in Examples 2 and 6 describedherein, a 4:2 (hypermethylated:hypomethylated) mixture of two plasmids,which results in about 40% to 45% of CpG methylation, achieved the bestresult in treating Type I diabetes. Therefore, while the methylationlevel may need to be adjusted or optimized or different types oftreatment, the screening or assessing such level can be achieved usingart known methods.

Similar rationales may be applied to the use of methylated DNA (e.g.,plasmid) as described herein for gene therapy. For gene therapy, twoconsiderations may be made with respect to optimal or desiredmethylation level. First is whether the foreign DNA would elicit immuneresponse to host cell, as described above. Second is how the expressionlevel of the foreign gene would be affected by methylation. Often, forgene therapy, the expression of the foreign gene is controlled by atissue-specific promoter that is activated in specific tissues. Sometissue-specific promoters may be more sensitive to methylationregulation than others. Nagase et al., (Epigenetics: differential DNAmethylation in mammalian somatic tissues, FEBS Journal 275 (2008)1617-1623) discloses different levels of promoter methylation indifferent mammalian tissues. One may take into consideration differencesin promoter methylation in different tissues when determining a desiredmethylation level for gene therapy. Reyes-Sandoval et al. (CpGMethylation of a Plasmid Vector Results in Extended Transgene ProductExpression by Circumventing Induction of Immune Responses, MolecularTherapy Vol. 9, 246-261 (2004)) reports the use of a CpG-methylatedplasmid expression vector expressing the highly immunogenic glycoproteinof rabies virus in order to achieve prolonged transgene productexpression by circumventing immune recognition. Their data show thatmice inoculated with a CpG-methylated plasmid expression vector showdelayed clearance of transfected cells and fail to mount a strong immuneresponse to the transgene product. Gene transfer with a CpG methylatedplasmid resulted in a state of immunological low responsiveness to thetransgene product, which may facilitate readministration of thetransgene.

Accordingly, DNA molecules, such as plasmids, produced by the bacterialstrains described herein can be used for gene therapy. The plasmids usedin gene therapy may carry (i) an origin of replication, (ii) a markergene such as a gene for resistance to an antibiotic (kanamycin,ampicillin, and the like) and (iii) one or more transgenes withsequences necessary for their expression (enhancer(s), promoter(s),polyadenylation sequences, and the like).

In another aspect, the subject technology provides a method of adjustingmethylation level of a DNA plasmid for treating an autoimmune disease,wherein said DNA plasmid encodes an autoantigen or an antigenic fragmentthereof, and is produced by a bacterium described herein, comprising:(i) administering said DNA plasmid to a subject in need thereof; (ii)determining a therapeutic effect of said DNA plasmid; and (iii)adjusting a methylation level of said DNA based on the therapeuticeffect.

In another aspect, the subject technology provides a method of adjustingmethylation level of a DNA plasmid for treating a transplant recipient,wherein said DNA plasmid encodes a donor antigen or an antigenicfragment thereof, and is produced by a bacterium described herein,comprising: (i) administering said DNA plasmid to a subject in needthereof; (ii) determining a therapeutic effect of said DNA plasmid; and(iii) adjusting a methylation level of said DNA based on the therapeuticeffect.

In another aspect, the subject technology provides a method of adjustingmethylation level of a DNA plasmid for gene therapy, wherein said DNAplasmid encodes a therapeutic protein and is operably linked to apromoter, and is produced by a bacterium described herein, comprising:(i) administering said DNA plasmid to a subject in need thereof; (ii)determining a therapeutic effect of said DNA plasmid; and (iii)adjusting a methylation level of said DNA based on the therapeuticeffect.

In another aspect, the subject technology provides a method ofexpressing a protein of interest in a target cell or target tissue of asubject, comprising: administering a plasmid DNA to a subject in needthereof, wherein said plasmid DNA comprises a polynucleotide thatencodes a therapeutic protein, and is operably linked to a promoter,wherein said promoter can be activated in the target cell or targettissue, and wherein said DNA plasmid is produced by the bacteriumdescribed herein.

In another aspect, the subject technology provides a method of providinga DNA plasmid for expressing a protein of interest in a target cell ortarget tissue of a subject, comprising: (i) selecting a promoter thatcan be activated in the target cell or target tissue; (ii) determining amethylation level for said promoter, such that a transcriptionalrepression of the promoter by methylation is no more than 70%(preferably, no more than 60%, no more than 50% no more than 40% no morethan 30% no more than 20%, or no more than 10%); (iii) operably linkingsaid promoter to a polynucleotide sequence encoding said protein ofinterest to create a DNA plasmid; (iv) producing said DNA plasmid usinga bacterium described herein, according to the methylation leveldetermined in step (ii).

Generally, methylation of a promoter represses the activity or strengthof the promoter. Preferably, methylation level of the promoter isdetermined such that methylation represses the activity or strength ofthe promoter by no more than 50%, as compared to the activity orstrength of the promoter prior to methylation (that is, when themethylation level of the promoter is about 15% or less, preferably 10%or less). Alternatively or in addition, the methylation level of apromoter sequence is about 60% or less, about 55% or less, about 50% orless, about 45% or less, about 40% or less, about 35% or less, about 30%or less, about 25% or less, or about 20% or less, such that the activityor strength of the promoter is not significantly repressed, and theprotein of interest is expressed at a sufficient level. The activity orstrength of a methylated promoter vs. a corresponding unmethylated(hypomethylated) promoter can be compared, for example, by comparing thequantities of mRNAs transcribed from an operably linked coding sequence.

In one embodiment, the subject technology includes a kit for preparing aplasmid for use as a DNA vaccine or a recombinant gene therapy agentcomprising: (a) a bacterium having chromosomal DNA comprising anengineered methylase gene controlled by a constitutive promoter stablyincorporated into the chromosomal DNA; and (b) instructions for use inexpressing a plasmid encoding a gene of interest. In variousembodiments, the bacterium comprises E. coli.

6. Polynucleotides Encoding a Pro-Apoptotic Protein

In one aspect, the invention provides a polynucleotide that comprises asequence that encodes a pro-apoptotic protein, or a functional fragmentthereof, and wherein about 30% to about 60% of the CpG dinucleotides ofthe polynucleotide are methylated.

In another aspect, the invention provides a polynucleotide thatcomprises a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, and wherein the methylation level of CpGdinucleotides of the polynucleotide is about 2 fold to about 4 foldhigher as compared to the average methylation level of CpG dinucleotidesin a wild type Escherichia coli (E. coli) genome.

A. Methylation Levels

DNA methylation in vertebrates typically occurs at CpG sites. Thismethylation results in the conversion of the cytosine to5-methylcytosine. The formation of Me-CpG is catalyzed by the enzyme DNAmethyltransferase. Between 60% and 90% of all CpGs are methylated inmammals. Human DNA has about 80%-90% of CpG sites methylated.

Bacterial DNA contains low levels of methylated CpG dinucleotidescompared to mammalian DNA. The mammalian immune system uses this featureof bacterial DNA as a signal to identify foreign DNA and respond tothreats by bacterial pathogens.

Therefore, the polynucleotide described herein is preferablyhypermethylated to avoid being recognized as a bacterial pathogen whenadministered to a mammalian subject. For example, at least about 70%, atleast about 65%, at least about 60%, at least about 55%, at least about50%, at least about 45%, at least about 40%, at least about 35%, atleast about 30%, from about 30% to about 70%, from about 30% to about65%, from about 30% to about 60%, from about 30% to about 55%, fromabout 30% to about 50%, from about 30% to about 45%, from about 30% toabout 40%, from about 25% to about 60%, from about 25% to about 55%,from about 25% to about 50%, of the CpG dinucleotides of thepolynucleotide are methylated.

Alternatively, the methylation level of the polynucleotide can bedetermined by comparing the overall CpG dinucleotides methylation levelagainst a baseline. A suitable baseline methylation level is the averageCpG dinucleotides methylation level of a bacterial DNA (e.g., bacterialgenome) from a wild type bacterium (e.g., an Escherichia coli (E. coli).For example, methylation level of CpG dinucleotides of thepolynucleotide can be at least about 1.5 fold, at least about 2 fold, atleast about 2.5 fold, at least about 3 fold, at least about 3.5 fold, atleast about 4 fold, at least about 4.5 fold, at least about 5 fold, atleast about 6 fold, from about 1.5 fold to about 6 fold, from about 2fold to about 5 fold, or from about 2 fold to about 4 fold, higher ascompared to the average methylation level of CpG dinucleotides in a wildtype Escherichia coli (E. coli) genome, or the average methylation levelof CpG dinucleotides in an episomal DNA, such as a plasmid from a wildtype E. coli. The average methylation level of CpG dinucleotides in wildtype bacterial strains are generally known or ascertainable.

The methylation level of the polynucleotide described herein, or thebaseline average methylation level of wild type bacterium can also bedetermined using art known methods. For example, WO2011109529 disclosesmethods for detecting the prevalence of methylation of a DNA sequence ina DNA sample; see also, Wu et al., Statistical Quantification ofMethylation Levels by Next-Generation Sequencing, PLoS ONE 6(6): e21034.doi:10.1371/journal.pone.0021034.

Methods of making a polynyucleotide with a desired methylation level canbe achieved by multiple methods. For example, PCT Patent ApplicationPCT/US12/56761 discloses a bacterial strain comprising an engineeredpolynucleotide sequence encoding a methylase controlled by aconstitutive promoter, wherein said engineered polynucleotide is stablyincorporated into the chromosomal DNA of said bacterium. This bacterialstrain can be used to produce polynucleotides that have a desiredmethylation level. For example, levels of methylation can be adjusted bymodulating the expression level of methylase in the bacterial host (forexample, by using promoters of different strength). In vitro methylationmay also be used. See, e.g., Wyngaert et al., Genomic Imprinting:Methods and Protocols, at Chapter 18, In Vitro Methylation ofPredetermined Regions in Recombinant DNA Constructs 243-250, DOI:10.1385/1-59259-2,1-2:243.

B. Pro-Apoptotic Proteins

Proapoptotic proteins refers to proteins that are able to activatedirectly or indirectly the mechanisms of cell death by apoptosis.

Suitable pro-apoptotic proteins include, e.g., Bax, Bak, Bim, Puma, Bad,Bik, Noxa, Bmf, Hrk, Bid, FAS, a caspase mutant (a modified caspase), asurvivin mutant (a modified survivin protein), Death Receptor 4 (DR4),Death Receptor 5 (DR5), a FAS receptor, and a Tumor Necrosis FactorReceptor. A functional fragment of any one of these pro-apoptoticproteins is also encompassed.

Exemplary pro-apoptotic proteins described herein have beencharacterized.

Members of the Bcl-2 family represent some of the most well-definedregulators of apoptosis pathway. Some members of the Bcl-2 family,including Bcl-2, Bcl-XL, Ced-9, Bcl-w and so forth, promote cellsurvival, while other members including Bax, Bcl-Xs, Bad, Bak, Bid, Bikand Bim have been shown to potentiate apoptosis (Adams and Cory, 1998).Biological functions of the Bcl-2 family members include dimer formation(Oltvai et al., J. Bcl-2 heterodimerizes in vivo with a conservedhomolog, Bax, that accelerates programmed cell death. Cell, 74: 609-619,1993.), protease activation (Chinnaiyan et al., a novel FAD D-homologousICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1)death-inducing signaling complex. Cell 1996; 85: 817-827), mitochondrialmembrane depolarization, generation of reactive oxygen intermediates(Hockenbery et al., Bcl-2 functions in an antioxidant pathway to preventapoptosis. Cell, 75: 241-251, 1993), regulation of calcium flux (Lam etal., Evidence that Bcl-2 represses apoptosis by regulating endoplasmicreticulum-associated Ca2+ fluxes. Proc. Natl. Acad. Sci. USA, 91:6569-6573, 1994; Huiling et al., Maintenance of calcium homeostasis inthe endoplasmic reticulum by Bcl-2. J. Cell Biol. 138: 1219-1228, 1997),and pore formation.

In the Bcl-2 family there is notable homology clustered within twoconserved regions: Bcl-2 homology domains 1 and 2 (BH1 and BH2) (Oltvaiet al., 1993; Boise et al., bcl-x, a bcl-2-related gene that functionsas a dominant regulator of apoptotic cell death. Cell. 1993 Aug. 27;74(4):597-608; Kozopas et al., MCL1, a gene expressed in programmedmyeloid cell differentiation, has sequence similarity to BCL2. Proc NatlAcad Sci USA. 1993 Apr. 15; 90(8):3516-20; Lin et al., Characterizationof A1, a novel hemopoietic-specific early-response gene with sequencesimilarity to bcl-2. J. Immunol. 1993 Aug. 15; 151(4):1979-88). Anotherconserved domain in Bax, distinct from BH1 and BH2, is termed BH3 andmediates cell death and protein binding functions. A subset of thepro-apoptotic proteins contains only the BH3 domain, implying that thisparticular domain may be uniquely important in the promotion ofapoptosis (Diaz et al., Dimerization properties of human BAD.Identification of a B H-3 domain and analysis of its binding to mutantBCL-2 and BCL-XL proteins. J. Biol. Chem. 1997 Dec. 5;272(49):30866-72).

Bax, a 21 kDa death-promoting member of the Bcl-2 family, was firstidentified as a protein that co-immunoprecipated with Bcl-2 fromdifferent cell lines (Oltvai et al., 1993). Overexpression of Baxaccelerates cell death in response to a wide range of cytotoxic results.Determination of the amino acid sequence of the Bax protein showed it tobe highly homologous to Bcl-2. The Bax gene consists of six exons andproduces alternative transcripts, the predominant form of which encodesa 1.0 kb mRNA and is designated Baxa. Like Bcl-2 and several othermembers of the Bcl-2 family, the Bax protein has highly conservedregions, BH1, BH2 and BH3 domains, and hydropathy analysis of thesequences of these proteins indicates the presence of a hydrophobictransmembrane segment at their C-terminal ends (Oltvai et al., 1993).Bax is widely expressed without any apparent tissue specificity.

A splice variant of Bax, Bax-alpha, has also been reported.

Bak is expressed in a wide variety of cell types and binds to the Bcl-2homologue Bcl-x2 in yeast. A domain in Bak was identified as bothnecessary and sufficient for cytotoxicity activity and binding toBcl-x1. Furthermore, sequences similar to this domain that are distinctfrom BH1 and BH2 have been identified in Bax and Bipl. This domain iscritical for mediating the function of multiple cell death-regulatoryproteins that interact with Bcl-2 family members.

Bad possesses the key amino acid motifs of BH1 and BH2 domains. Badlacks the classical C-terminal signal-anchor sequence responsible forthe integral membrane positions of other family members. Bad selectivelydimerizes with Bcl-X_(L) as well as Bcl-2, but not with Bax,BcI-Xs-Mcl1, A1 or itself. Bad reverses the death repressor activity ofBcl-X_(L), but not that of Bcl-2.

Bik, another member of the Bcl-2 family, interacts with the cellularsurvival-promoting proteins, Bcl-2 and Bcl-X_(L) as well as the viralsurvival-promoting proteins, Epstein Barr virus-BHRF1 and adenovirusE1B-19 kDa. In transient transfection assays, Bik promotes cell death ina manner similar to Bax and Bak, other pro-apoptotic members of theBcl-2 family. This pro-apoptosis activity of Bik can be suppressed bycoexpression of Bcl-2, Bcl-X_(L), EBV-BHRF1 and EIB-19 kDa proteins,which suggests that Bik may be a common target for both cellular andviral anti-apoptotic proteins. While Bik does not contain overt homologyto the BH1 and BH2 conserved domains characteristic of the Bcl-2 family,it shares a 9 amino acid domain (BH3) with Bax and Bak, which may be acritical determinant for the death-promoting activity of these proteins.

PUMA (p53 upregulated modulator of apoptosis) is a target for activationby p53. A gene encodes two BH3 domain-containing proteins (PUMA-alphaand PUMA-beta) that are induced in cells following p53 activation.PUMA-alpha and PUMA-beta show similar activities; they bind to Bcl-2,localize to the mitochondria to induce cytochrome c release, andactivate the rapid induction of programmed cell death. Antisenseinhibition of PUMA expression reduced the apoptotic response to p53, andPUMA is likely to play a role in mediating p53-induced cell deaththrough the cytochrome c/Apaf-1-dependent pathway. See, e.g., Nakano etal., Mol Cell. 2001 March; 7(3):683-94.

The BH3-only protein Bim is a pro-apoptotic protein that inducesBax/Bak-oligomerization on mitochondria. See, e.g., Gogada et al., BIM,a proapoptotic protein, upregulated via transcription factorE2F1-dependent mechanism, functions as a prosurvival molecule in cancer,doi:10.1074/jbc.M112.386102jbc.M112.386102.

Activation of the intrinsic apoptotic pathway by p53 often requires thetranscription of the proapoptotic Bcl-2 proteins Noxa, Puma, or both. Ithas been reported that Noxa is required for particulate matter-inducedcell death and lung inflammation.

Bmf is a proapoptotic BH3-only protein regulated by interaction with themyosin V actin motor complex, activated by anoikis.

BH3-only family member HRK (also known as DP5) is involved in apoptosisregulation. Hrk gene expression was found to be restricted to cells andtissues of the central and peripheral nervous systems.

The BH3 interacting-domain death agonist, or BID, gene is apro-apoptotic member of the Bcl-2 protein family. In response toapoptotic signaling, BID interacts with another Bcl-2 family protein,Bax, leading to the insertion of Bax into organelle membranes, primarilythe outer mitochondrial membrane. Bax is believed to interact with, andinduce the opening of the mitochondrial voltage-dependent anion channel,VDAC. Alternatively, growing evidence suggest that activated Bax and/orBak form an oligomeric pore, MAC in the outer membrane. This results inthe release of cytochrome c and other pro-apoptotic factors (such asSMAC/DIABLO) from the mitochondria, often referred to as mitochondrialouter membrane permeabilization, leading to activation of caspases. Thisdefines BID as a direct activator of Bax, a role common to some of thepro-apoptotic Bcl-2 proteins containing only the BH3 domain.

Fas ligand (FasL or CD95L) is a type-II transmembrane protein thatbelongs to the tumor necrosis factor (TNF) family. Its binding with itsreceptor induces apoptosis. Fas ligand/receptor interactions play animportant role in the regulation of the immune system and theprogression of cancer.

The FAS receptor (FasR), also known as apoptosis antigen 1 (APO-1 orAPT), cluster of differentiation 95 (CD95) or tumor necrosis factorreceptor superfamily member 6 (TNFRSF6) is a protein that in humans isencoded by the TNFRSF6 gene. The Fas receptor is a death receptor on thesurface of cells that leads to programmed cell death (apoptosis). It isone of two apoptosis pathways, the other being the mitochondrialpathway. FasR is located on chromosome 10 in humans and 19 in mice.Similar sequences related by evolution (orthologs) are found in mostmammals.

Caspases, or cysteine-aspartic proteases or cysteine-dependentaspartate-directed proteases are a family of cysteine proteases thatplay essential roles in apoptosis (programmed cell death), necrosis, andinflammation. Twelve caspases have been identified in humans. There aretwo types of apoptotic caspases: initiator (apical) caspases andeffector (executioner) caspases. Initiator caspases (e.g., CASP2, CASP8,CASP9, and CASP10) cleave inactive pro-forms of effector caspases,thereby activating them. Effector caspases (e.g., CASP3, CASP6, CASP7)in turn cleave other protein substrates within the cell, to trigger theapoptotic process. The initiation of this cascade reaction is regulatedby caspase inhibitors. CASP4 and CASP5, which are overexpressed in somecases of vitiligo and associated autoimmune diseases caused by NALP1variants, are not currently classified as initiator or effector in MeSH,because they are inflammatory enzymes that, in concert with CASP1, areinvolved in T-cell maturation. Also contemplated herein are mutants ofcapspases that retains the pro-apoptotic activity.

Survivin, also called baculoviral inhibitor of apoptosisrepeat-containing 5 or BIRC5, is a protein that, in humans, is encodedby the BIRC5 gene. NCBI Reference

Sequence: NG_029069.1. Survivin is a member of the inhibitor ofapoptosis (IAP) family. The survivin protein functions to inhibitcaspase activation, thereby leading to negative regulation of apoptosisor programmed cell death. Contemplated herein are mutants of Survivinthat abolish the anti-apopotitc activity.

Death Receptor 4 (DR4) is a protein on the surface of certain cells thatbinds another protein called TRAIL, which may kill some cancer cells. Anincrease in the amount or activity of death receptor 4 on cancer cellsmay kill more cells. Also called DR4, TRAIL receptor 1, TRAIL-R1, andtumor necrosis factor receptor superfamily member 10A.

Tumor necrosis factor receptor superfamily, member 10b, is also knownas: DR5, CD262, KILLER, TRICK2, TRICKB, ZTNFR9, TRAILR2, TRICK2A,TRICK2B, TRAIL-R2, KILLER/DR5. The protein is a member of theTNF-receptor superfamily, and contains an intracellular death domain.This receptor can be activated by tumor necrosis factor-relatedapoptosis inducing ligand (TNFSF10/TRAIL/APO-2L), and transducesapoptosis signal. Mice have a homologous gene, tnfrsf10b, that has beenessential in the elucidation of the function of this gene in humans.Studies with FADD-deficient mice suggested that FADD, a death domaincontaining adaptor protein, is required for the apoptosis mediated bythis protein.

A tumor necrosis factor receptor (TNFR), or death receptor, is atrimeric cytokine receptor that binds tumor necrosis factors (TNF). Thereceptor cooperates with an adaptor protein (such as TRADD, TRAF, RIP),which is important in determining the outcome of the response (e.g.apoptosis, inflammation).

Pro-apoptotic fragments of the pro-apoptotic proteins described hereinmay also be used. A pro-apoptotic fragment of a pro-apoptotic comprisesa portion, but no the full-length sequence of the pro-apoptotic, whileretaining the pro-apoptotic activity.

The pro-apoptotic protein or fragments thereof can be a naturallyoccurring protein which has pro-apoptotic activity, or an active variantof a naturally occurring protein.

As used herein, “active variants” refers to variant peptides whichretain pro-apoptotic activity. An active variant differs in amino acidsequence from a reference pro-apoptotic protein but retainspro-apoptotic activity. Active variants of pro-apoptotic proteins orfragments thereof include naturally occurring variants (e.g., allelicforms) and variants which are not known to occur naturally.

In general, pro-apoptotic fragments can contain the functional domain ofthe pro-apoptotic proteins.

Generally, differences are limited so that the sequences of thereference polypeptide and the active variant are closely similar overalland, in many regions, identical. An active variant of a pro-apoptoticprotein or fragment thereof and a reference pro-apoptotic protein orfragment thereof can differ in amino acid sequence by one or more aminoacid substitutions, additions, deletions, truncations, fusions or anycombination thereof. Preferably, amino acid substitutions areconservative substitutions. A conservative amino acid substitutionrefers to the replacement of a first amino acid by a second amino acidthat has chemical and/or physical properties (e.g., charge, structure,polarity, hydrophobicity/hydrophilicity) which are similar to those ofthe first amino acid. Conservative substitutions include replacement ofone amino acid by another within the following groups: lysine (K),arginine (R) and histidine (H); aspartate (D) and glutamate (E);asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y),K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I),proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine(C) and glycine (G); F, W and Y; C, S and T.

Active variants of pro-apoptotic proteins or fragments thereof can beprepared using suitable methods, for example, by direct synthesis,mutagenesis (e.g., site directed mutagenesis, scanning mutagenesis) andother methods of recombinant DNA technology. Active variants can beidentified and/or selected using a suitable apoptosis assay.

Fusion proteins comprising a pro-apoptotic protein or a fragment thereofare also contemplated. A fusion protein may encompass a polypeptidecomprising a pro-apoptotic protein, a fragment thereof, or an activevariant thereof as a first moiety, linked via a covalent bond (e.g., apeptide bond) to a second moiety (a fusion partner) not occurring in thepro-apoptotic protein as found in nature. Fusion proteins can beprepared using suitable methods, for example, by direct synthesis,recombinant DNA technology, etc.

The invention also relates to nucleic acids encoding a pro-apoptoticprotein, a fragment thereof, or a variant thereof.

C. Autoantigens.

Optionally, the compositions described herein may comprises anautoantigen. The autoantigen can be encoded by the same polynucleotide,or by a second polynucleotide. Preferably, the autoantigen is present inskin or in hair follicles.

7. Delivery of Polynucleotides

Being a negatively charged, high molecular weight molecule,polynucleotides have difficulties in passing spontaneously through thephospholipid cell membranes. Various vectors are hence used in order topermit gene transfer, including viral vectors on the one hand, naturalor synthetic chemical and/or biochemical vectors on the other hand.

Viral vectors (retroviruses, adenoviruses, adeno-associated viruses,etc.) are very effective, in particular for passing through themembranes, but present a number of risks, such as pathogenicity,recombination, replication, immunogenicity, and the like. Chemicaland/or biochemical vectors enable these risks to be avoided. They are,for example, cations (calcium phosphate, DEAE-dextran, and the like.)which act by forming precipitates with DNA, which precipitates can thenbe “phagocytosed” by the cells. They can also be liposomes in which theDNA is incorporated and which fuse with the plasma membrane. Syntheticgene transfer vectors are generally cationic lipids or polymers whichcomplex DNA can form therewith a particle carrying positive surfacecharges. These particles are capable of interacting with the negativecharges of the cell membrane and then of crossing the latter. Examplesof such vectors can include dioctadecylamidoglycylspermine (DOGS,Transfectam™) or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA, Lipofectin™). Chimeric proteins have also beendeveloped: they consist of a polycationic portion which condenses theDNA, linked to a ligand which binds to a membrane receptor and givesrise to the complex in the cells by endocytosis. It is thustheoretically possible to “target” a tissue or certain cell populationsin order to improve the in vivo bioavailability of the transferred gene.

Viral vector can be delivered to a host cell in the form of arecombinant virus. Recombinant viral vector can be packaged into viralcoats or capsids by any suitable procedure, for example, by transfectingthe recombinant viral vector into a packaging cell line. Any suitablepackaging cell line can be used to generate recombinant virus. Suitablepackaging lines for retroviruses include derivatives of PA317 cells, ψ-2cells, CRE cells, CRIP cells, E-86-GP cells, and 293GP cells. Line 293cells can be used for adenoviruses and adeno-associated viruses.Neuroblastoma cells can be used for herpes simplex virus, e.g. herpessimplex virus type 1. Sometimes, a helper virus (which provides missingproteins for production of new virions) may be needed to produce arecombinant virus described herein.

Nucleic acid molecules encoding a pro-apoptotic protein, or a fragmentthereof can also be delivered using a non-viral based nucleic aciddelivery system. For example, methods of delivering a nucleic acid to atarget cell have been described in U.S. Pat. Nos. 6,413,942, 6,214,804,5,580,859, 5,589,466, 5,763,270 and 5,693,622.

Nucleic acid molecules described herein can be packaged in liposomesprior to delivery to a subject or to cells, as described in U.S. Pat.Nos. 5,580,859, 5,549,127, 5,264,618, 5,703,055. For a review of the useof liposomes as carriers for delivery of nucleic acids, see, Hug andSleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger et al.(1983) in Methods of Enzymology Vol. 101, pp. 512-27; de Lima et al.(2003) Current Medicinal Chemistry, Volume 10(14): 1221-31.Representative liposomes include, but not limited to cationic liposomes,optionally coated with polyethylene glycol (PEG) to reduce non-specificbinding of serum proteins and to prolong circulation time. See Koning etal., 1999; Nam et al., 1999; and Kirpotin et al., 1997.Temperature-sensitive liposomes can also be used, for exampleTHERMOSOMES™ as disclosed in U.S. Pat. No. 6,200,598. The use ofvector-liposome complexes has been described in U.S. Pat. No. 5,851,818.Liposomes can be prepared by any of a variety of techniques that areknown in the art. See e.g., Betageri et al., 1993; Gregoriadis, 1993;Janoff, 1999; Lasic & Martin, 1995; Nabel, 1997; and U.S. Pat. Nos.4,235,871; 4,551,482; 6,197,333; and 6,132,766.

Nucleic acids can also be delivered in cochleate lipid compositionssimilar to those described by Papahadjopoulos et al. (1975) Biochem.Biophys. Acta. 394:483-491. See also U.S. Pat. Nos. 4,663,161 and4,871,488. For example, a plasmid vector may be complexed withLipofectamine 2000. Wang et al. (2005) Mol. Therapy 12(2):314-320.

Biolistic delivery systems employing particulate carriers such as goldand tungsten may also be used to deliver nucleic acids (e.g.,recombinant viral vectors and non-viral vectors). The particles arecoated with the vector and accelerated to high velocity, generally underreduced pressure, using a gun powder discharge from a “gene gun.” See,e.g., U.S. Pat. Nos. 4,945,050, 5,036,006, 5,100,792, 5,179,022,5,371,015, and 5,478,744.

A wide variety of other methods can be used to deliver the nucleic acidsdescribed herein. Such methods include DEAE dextran-mediatedtransfection, calcium phosphate precipitation, polylysine- orpolyornithine-mediated transfection, or precipitation using otherinsoluble inorganic salts, such as strontium phosphate, aluminumsilicates including bentonite and kaolin, chromic oxide, magnesiumsilicate, talc, and the like. Other useful methods of transfectioninclude electroporation, sonoporation, protoplast fusion, peptoiddelivery, or microinjection. See, e.g., Sambrook et al (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York,for a discussion of techniques for transforming cells of interest; andFelgner, P. L. (1990) Advanced Drug Delivery Reviews 5:163-87, for areview of delivery systems useful for gene transfer. Exemplary methodsof delivering DNA using electroporation are described in U.S. Pat. Nos.6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No.2002/0146831, and International Publication No. WO 00/45823.

8. Pharmaceutical Compositions and Methods of Treatment

One aspect of the subject technology describes methods to treat hairloss, the use of the polynucleotides described herein in therapy, andthe use of the polynucleotides described herein in the manufacture of amedicament for therapy. The term treatment including prophylactictreatment in the absence of a symptom, as well as treatment thatalleviated a disease, or symptoms of a disease.

The subject technology provides a pharmaceutical composition comprisinga polynucleotide described herein. Optionally, a delivery system fordelivering the nucleic acid (e.g., liposomes) may be provided. Thedelivery system can be co-formulated with the nucleic acid into apharmaceutical composition, or supplied separately (e.g., in a separatecontainer in a kit). If provided separately, the delivery system may bemixed with the nucleic acid prior to administration.

Polynucleotides disclosed herein are generally formulated in acomposition suitable for in vivo administration. Such compositionsgenerally include a carrier that can is acceptable for formulating andadministering the agent to a subject. Such acceptable carriers are wellknown in the art and include, for example, aqueous solutions such aswater or physiologically buffered saline or other solvents or vehiclessuch as glycols, glycerol, oils such as olive oil or injectable organicesters. An acceptable carrier can contain physiologically acceptablecompounds that act, for example, to stabilize or to increase theabsorption of the conjugate. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art would know that the choice of anacceptable carrier, including a physiologically acceptable compound,depends, for example, on the physico-chemical characteristics of thetherapeutic agent and on the route of administration of the composition,which can be, for example, orally or parenterally such as intravenously,and by injection, intubation, or other such method known in the art. Thepharmaceutical composition also can contain a second reagent such as adiagnostic reagent, nutritional substance, toxin, or therapeutic agent,for example, a cancer chemotherapeutic agent.

Polynucleotides described herein can be incorporated within anencapsulating material such as into an oil-in-water emulsion, amicroemulsion, micelle, mixed micelle, liposome, microsphere or otherpolymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol.1 (CRC Press, Boca Raton, Fla. 1984); Fraley, et al., Trends Biochem.Sci., 6:77 (1981)). Liposomes, for example, which consist ofphospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister. “Stealth” liposomes (see, for example, U.S. Pat. Nos.5,882,679; 5,395,619; and 5,225,212) are an example of suchencapsulating materials particularly useful for preparing a compositionuseful in a method of the invention, and other “masked” liposomessimilarly can be used, such liposomes extending the time that thetherapeutic agent remain in the circulation. Cationic liposomes, forexample, also can be modified with specific receptors or ligands(Morishita et al., J. Clin. Invest., 91:2580-2585 (1993), which isincorporated herein by reference). In addition, a polynucleotide can beintroduced into a cell using, for example, adenovirus-polylysine DNAcomplexes (see, for example, Michael et al., J. Biol. Chem.268:6866-6869 (1993)).

In one aspect, the subject technology provides a method of treatingAlopecia Areata, comprising: administering to a subject in need thereofan effective amount of a polynucleotide, wherein said polynucleotidecomprises a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, and wherein about 30% to about 60% of theCpG dinucleotides of the polynucleotide are methylated.

In another aspect, the subject technology provides a method of treatingAlopecia Areata, comprising: administering to a subject in need thereofan effective amount of a polynucleotide, wherein said polynucleotidecomprises a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, and wherein the methylation level of CpGdinucleotides of the polynucleotide is about 2 fold to about 4 foldhigher as compared to the average methylation level of CpG dinucleotidesin a wild type Escherichia coli (E. coli) genome.

The subject can be an alopecia subject having a history of, orcoexisting, allergic disease or autoimmune disease.

Alopecia areata can be normal alopecia areata, alopecia totalis,alopecia universalis, or alopecia ophiasis.

Compositions described herein may also be used to treat white hair,vellus hair formation, or an agent for inhibiting seborrheic scalp orthe occurrence of dandruff.

In one aspect, the subject technology provides a method of stimulatinggrowth of a hair shaft from a hair follicle, comprising: administeringto a subject in need thereof an effective amount of a polynucleotide,wherein said polynucleotide comprises a sequence that encodes apro-apoptotic protein, or a functional fragment thereof, and whereinabout 30% to about 60% of the CpG dinucleotides of the polynucleotideare methylated.

In one aspect, the subject technology provides a method of stimulatinggrowth of a hair shaft from a hair follicle, comprising: administeringto a subject in need thereof an effective amount of a polynucleotide,wherein said polynucleotide comprises a sequence that encodes apro-apoptotic protein, or a functional fragment thereof, and wherein themethylation level of CpG dinucleotides of the polynucleotide is about 2fold to about 4 fold higher as compared to the average methylation levelof CpG dinucleotides in a wild type Escherichia coli (E. coli) genome.

In one aspect, the subject technology provides a method of treating hairloss, wherein said hair loss is attributable to an autoimmune disease,comprising: administering to a subject in need thereof an effectiveamount of a polynucleotide, wherein said polynucleotide comprises asequence that encodes a pro-apoptotic protein, or a functional fragmentthereof, and wherein about 30% to about 60% of the CpG dinucleotides ofthe polynucleotide are methylated.

In one aspect, the subject technology provides a method of stimulatinggrowth of a hair shaft from a hair follicle, comprising: administeringto a subject in need thereof an effective amount of a polynucleotide,wherein said polynucleotide comprises a sequence that encodes apro-apoptotic protein, or a functional fragment thereof, and wherein themethylation level of CpG dinucleotides of the polynucleotide is about 2fold to about 4 fold higher as compared to the average methylation levelof CpG dinucleotides in a wild type Escherichia coli (E. coli) genome.

In many instances, hair loss can be attributed to an autoimmunecondition. Exemplary autoimmune diseases that could result in hair lossinclude, e.g., chronic discoid lupus erythematosus, localizedscleroderma, pemphigus, pemphigoid, herpes gestationis, linear IgAbullous dermatosis, acquired epidermolysis bullosa, vitiligo, Sutton'snevus, an autoimmune thyroid disease, systemic lupus erythematosus,rheumatoid arthritis, or myasthenia gravis

Androgenetic alopecia can also results in hair loss. Androgeneticalopecia can be in a male or androgenetic alopecia in a female.

An effective amount is administered to a subject in need thereof. An“effective amount” or “therapeutically effective amount” is an amountthat is sufficient to achieve the desired therapeutic or prophylacticeffect under the conditions of administration, such as an amountsufficient to reduce/ameliorate symptoms of disease.

The route of administration of the composition containingpolynucleotides described herein will depend, in part, on the chemicalstructure of the molecule. Polypeptides and polynucleotides, forexample, are not particularly useful when administered orally becausethey can be degraded in the digestive tract. However, methods forchemically modifying polypeptides, for example, to render them lesssusceptible to degradation by endogenous proteases or more absorbablethrough the alimentary tract are disclosed herein or otherwise known inthe art (see, for example, Blondelle et al., supra, 1995; Ecker andCrook, supra, 1995). In addition, a polypeptide can be prepared usingD-amino acids, or can contain one or more domains based onpeptidomimetics, which are organic molecules that mimic the structure ofa domain; or based on a peptoid such as a vinylogous peptoid.

A composition as disclosed herein can be administered to an individualby various routes including, for example, orally or parenterally, suchas intravenously, intramuscularly, subcutaneously, intraorbitally,intracapsularly, intraperitoneally, intrarectally, intracistemally or bypassive or facilitated absorption through the skin using, for example, askin patch or transdermal iontophoresis, respectively. Furthermore, thecomposition can be administered by injection, intubation, orally ortopically, the latter of which can be passive, for example, by directapplication of an ointment, or active, for example, using a nasal sprayor inhalant, in which case one component of the composition is anappropriate propellant. A pharmaceutical composition also can beadministered to the site of a pathologic condition, for example, to thescalp where hair loss is occurring, or is likely to occur.

Preferred route of administration includes at least one ofsubcutaneously, intradermally, or transdermally. The compositions can beadministered to the hair loss site, e.g., the frontal region or thecrown.

The total amount of the polynucleotide to be administered in practicinga method of the invention can be administered to a subject as a singledose, either as a bolus or by infusion over a relatively short period oftime, or can be administered using a fractionated treatment protocol, inwhich multiple doses are administered over a prolonged period of time.One skilled in the art would know that the amount of the composition totreat a pathologic condition in a subject depends on many factorsincluding the age and general health of the subject as well as the routeof administration and the number of treatments to be administered. Inview of these factors, the skilled artisan would adjust the particulardose as necessary. In general, the formulation of the composition andthe routes and frequency of administration are determined, initially,using Phase I and Phase II clinical trials.

In various embodiments, when the method comprises administering one ormore than one immunosuppressant agent(s), the one or more than oneimmunosuppressant agent(s) can be administered simultaneously,separately or sequentially.

In various embodiments, the one or more than one immunosuppressantagent(s) may be selected from the group consisting of corticosteroids,glucocorticoids. cyclophosphamide, 6-mercaptopurine (6-MP), azathioprine(AZA), methotrexate cyclosporine, mycophenolate mofetil (MMF),mycophenolic acid (MPA), tacrolimus (FK506), sirolimus ([SRL]rapamycin), everolimus (Certican), mizoribine, leflunomide,deoxyspergualin, brequinar, azodicarbonamide, vitamin D analogs, such asMC1288 and bisindolylmaleimide VIII, antilymphocyte globulin,antithymocyte globulin (ATG), anti-CD3 monoclonal antibodies,(Muromonab-CD3, Orthoclone OKT3), anti-interleukin (IL)-2 receptor(anti-CD25) antibodies, (Daclizumab, Zenapax, basiliximab, Simulect),anti-CD52 antibodies, (Alemtuzumab, Campath-1H), anti-CD20 antibodies(Rituximab, Rituxan), anti-tumor necrosis factor (TNF) reagents(Infliximab, Remicade, Adalimumab, Humira), LFA-1 inhibitors(Efalizumab, Raptiva), and the like.

In one aspect, the subject technology provides a polynucleotidecomprising a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, wherein about 30% to about 60% of the CpGdinucleotides of the polynucleotide are methylated for the use oftreating alopecia areata.

In one aspect, the subject technology provides a polynucleotidecomprising a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, wherein the methylation level of CpGdinucleotides of the polynucleotide is about 2 fold to about 4 foldhigher as compared to the average methylation level of CpG dinucleotidesin a wild type Escherichia coli (E. coli) genome for the use of treatingalopecia areata.

In one aspect, the subject technology provides a polynucleotidecomprising a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, wherein about 30% to about 60% of the CpGdinucleotides of the polynucleotide are methylated for the use ofstimulating growth of a hair shaft from a hair follicle.

In one aspect, the subject technology provides a polynucleotidecomprising a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, wherein the methylation level of CpGdinucleotides of the polynucleotide is about 2 fold to about 4 foldhigher as compared to the average methylation level of CpG dinucleotidesin a wild type Escherichia coli (E. coli) genome for the use ofstimulating growth of a hair shaft from a hair follicle.

In one aspect, the subject technology provides a polynucleotidecomprising a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, wherein about 30% to about 60% of the CpGdinucleotides of the polynucleotide are methylated for the use oftreating hair loss, wherein said hair loss is attributable to anautoimmune disease.

In one aspect, the subject technology provides a polynucleotidecomprising a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof, wherein the methylation level of CpGdinucleotides of the polynucleotide is about 2 fold to about 4 foldhigher as compared to the average methylation level of CpG dinucleotidesin a wild type Escherichia coli (E. coli) genome for the use of treatinghair loss, wherein said hair loss is attributable to an autoimmunedisease.

9. Other Methods of Treatment

One aspect of the subject technology describes methods to enhance thepotency of DNA vaccines for the treatment of various immune-mediatedinflammatory disorders, including rejection of solid organ transplants,graft versus host disease, host versus graft disease, autoimmunehepatitis, vitiligo, diabetes mellitus type 1, Addison's Disease,Graves' disease, Hashimoto's thyroiditis, multiple sclerosis,polymyalgia rheumatica, Reiter's syndrome, Crohn's disease,Goodpasture's syndrome, Gullain-Barré syndrome, lupus nephritis,rheumatoid arthritis, systemic lupus erythematosus, Wegener'sgranulomatosis, celiac disease, dermatomyositis, eosinophilic fasciitis,idiopathic thrombocytopenic purpura, Miller-Fisher syndrome, myastheniagravis, pemphigus vulgaris, pernicious anaemia, polymyositis, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis,Sjögren's syndrome, and the like.

As described herein, different levels of DNA methylation may be desiredfor different applications. Levels of methylation can be modulated atleast by two ways. First, it can be achieved by modulating theexpression level of methylase (e.g., by using promoters of differentstrength). Second, it may be achieved by mixing hypermethylated DNA(e.g., DNA in which about 50% of the CpG dinucleotides are methylated)with hypomethylated DNA (e.g., DNA in which about 10% to about 15% ofthe CpG dinucleotides are methylated), and adjusting ratios of thehypermethylated DNA to hypomethylated DNA.

In one aspect, the subject technology provides a method for treating anautoimmune disease, comprising administering to a subject in needthereof a therapeutically effective amount of: (a) a firstpolynucleotide comprising a sequence that encodes an autoantigen, or anantigenic fragment thereof, wherein at least about 70%, at least about65%, at least about 60%, at least about 55%, at least about 50%, atleast about 45%, at least about 40%, at least about 35%, at least about30%, or at least about 25% of the CpG dinucleotides of said firstpolynucleotide are methylated; and (b) a second polynucleotidecomprising a sequence that encodes a pro-apoptotic protein, or afunctional fragment thereof; wherein about 10% or less, about 5% orless, about 3% or less, or about 1% or less, of the CpG dinucleotides ofsaid second polynucleotide are methylated.

In various embodiments, said first polynucleotide and secondpolynucleotide are administered at a ratio of from about 10:1 to about1:10 (μg/μg). In exemplary embodiments, the ratios are from about 4:1 toabout 4:2 (μg/μg). Other suitable ratios include, e.g., about 10:1,about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, from about 5:1to about 1:5, from about 5:1 to about 1:3, from about 5:1 to about 1:1,from about 5:1 to about 2:1, or from about 4:1 to about 1:1, etc.

Optimal or preferred range of ratios of the two DNAs may be determinedby a skilled person using known screening methods. For example, asexemplified herein, different ratios of hypermethylated sGAD-codingsequence (msGAD) to hypomethylated BAX-coding sequences (BAX) werescreened using NOD mice. It was discovered, based on percentage ofdiabetic incidence, that a mixture at 4:2 ratio provides superiorresults as compared to 4:1 ratio. Similar screening assays can beapplied to assess and optimize the ratios of hypermethylated DNA andhypomethylated DNA.

In another aspect, the subject technology provides a method for treatingan autoimmune disease, comprising administering to a subject in needthereof a therapeutically effective amount of: (a) a firstpolynucleotide comprising a sequence that encodes an autoantigen, or anantigenic fragment thereof; and (b) a second polynucleotide comprising asequence that encodes a pro-apoptotic protein, or a functional fragmentthereof; wherein from about 10% to about 50% of the CpG dinucleotides ofsaid first and second polynucleotide are methylated. Preferably, fromabout 25% to about 45%, from about 30% to about 45%, from about 35% toabout 45%, or from about 40% to about 45% of the CpG dinucleotides ofsaid first and second polynucleotide are methylated.

The first polynucleotide and the second polynucleotide may be the samepolynucleotide (i.e., a polynucleotide encoding both proteins), or maybe different (i.e., two polynucleotides, one encoding the autoantigen,and the other encoding the pro-apoptotic protein). If the two proteinsare encoded by two different polypeptides, the methylation levels of thetwo polynucleotide can be different, as long the as the combined CpGmethylation level is within a desired range. For example, if about 42%of CpG methylation is desired, one can mix the first polynucleotide (atabout 50% CpG methylation level) with the second polynucleotide (atabout 10% CpG methylation level), at about 4:1 ratio, to achieve a finalCpG methylation level at about 42%.

Suitable autoantigens for certain autoimmune diseases are described inthe Table above. Suitable pro-apoptotic proteins are also describedabove.

In certain embodiments, the autoimmune disease is Type I diabetes. Incertain embodiments, the autoantigens is said autoantigen is glutamicacid decarboxylase (GAD), a secreted form of GAD (sGAD), or anauto-antigenic fragment thereof. Nucleic acid sequences encoding humanGAD, and a secreted form of human GAD, are provided herein as SEQ IDNOS: 2 and 3, respectively. In certain embodiments, the pro-apoptoticprotein is BAX, or a functional fragment thereof.

In various embodiments, when the method comprises administering one ormore than one immunosuppressant agent(s), the one or more than oneimmunosuppressant agent(s) can be administered simultaneously,separately or sequentially.

In various embodiments, the one or more than one immunosuppressantagent(s) may be selected from the group consisting of corticosteroids,glucocorticoids. cyclophosphamide, 6-mercaptopurine (6-MP), azathioprine(AZA), methotrexate cyclosporine, mycophenolate mofetil (MMF),mycophenolic acid (MPA), tacrolimus (FK506), sirolimus ([SRL]rapamycin), everolimus (Certican), mizoribine, leflunomide,deoxyspergualin, brequinar, azodicarbonamide, vitamin D analogs, such asMC1288 and bisindolylmaleimide VIII, antilymphocyte globulin,antithymocyte globulin (ATG), anti-CD3 monoclonal antibodies,(Muromonab-CD3, Orthoclone OKT3), anti-interleukin (IL)-2 receptor(anti-CD25) antibodies, (Daclizumab, Zenapax, basiliximab, Simulect),anti-CD52 antibodies, (Alemtuzumab, Campath-1H), anti-CD20 antibodies(Rituximab, Rituxan), anti-tumor necrosis factor (TNF) reagents(Infliximab, Remicade, Adalimumab, Humira), LFA-1 inhibitors(Efalizumab, Raptiva), and the like.

In one embodiment, the method further comprises, after administering theDNA vaccine, monitoring the recipient for rejection of the allograft oftransplant. In a preferred embodiment, the recipient is monitored forrejection of the allograft or transplant after tapering off ordiscontinuing the administration of immunosuppressant agent(s).

For administration to a subject, polynucleotides disclosed herein aregenerally formulated in a composition suitable for in vivoadministration. Such compositions generally include a carrier that canis acceptable for formulating and administering the agent to a subject.Such acceptable carriers are well known in the art and include, forexample, aqueous solutions such as water or physiologically bufferedsaline or other solvents or vehicles such as glycols, glycerol, oilssuch as olive oil or injectable organic esters. An acceptable carriercan contain physiologically acceptable compounds that act, for example,to stabilize or to increase the absorption of the conjugate. Suchphysiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. One skilled in the artwould know that the choice of an acceptable carrier, including aphysiologically acceptable compound, depends, for example, on thephysico-chemical characteristics of the therapeutic agent and on theroute of administration of the composition, which can be, for example,orally or parenterally such as intravenously, and by injection,intubation, or other such method known in the art. The pharmaceuticalcomposition also can contain a second reagent such as a diagnosticreagent, nutritional substance, toxin, or therapeutic agent, forexample, a cancer chemotherapeutic agent.

Polynucleotides described herein can be incorporated within anencapsulating material such as into an oil-in-water emulsion, amicroemulsion, micelle, mixed micelle, liposome, microsphere or otherpolymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol.1 (CRC Press, Boca Raton, Fla. 1984); Fraley, et al., Trends Biochem.Sci., 6:77 (1981)). Liposomes, for example, which consist ofphospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister. “Stealth” liposomes (see, for example, U.S. Pat. Nos.5,882,679; 5,395,619; and 5,225,212) are an example of suchencapsulating materials particularly useful for preparing a compositionuseful in a method of the invention, and other “masked” liposomessimilarly can be used, such liposomes extending the time that thetherapeutic agent remain in the circulation. Cationic liposomes, forexample, also can be modified with specific receptors or ligands(Morishita et al., J. Clin. Invest., 91:2580-2585 (1993), which isincorporated herein by reference). In addition, a polynucleotide can beintroduced into a cell using, for example, adenovirus-polylysine DNAcomplexes (see, for example, Michael et al., J. Biol. Chem.268:6866-6869 (1993)).

The composition can be formulated for oral formulation, such as atablet, or a solution or suspension form; or can comprise an admixturewith an organic or inorganic carrier or excipient suitable for enteralor parenteral applications, and can be compounded, for example, with theusual non-toxic, pharmaceutically acceptable carriers for tablets,pellets, capsules, suppositories, solutions, emulsions, suspensions, orother form suitable for use. The carriers, in addition to thosedisclosed above, can include glucose, lactose, mannose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, medium chainlength triglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening or coloring agents andperfumes can be used, for example a stabilizing dry agent such astriulose (see, for example, U.S. Pat. No. 5,314,695).

In one embodiment, the method further comprises administering a dose ofone or more than one immunosuppressant agent before, on the day of,and/or after engraftment or transplantation.

The dosages of the immunosuppressant agents will vary depending on theindividual to be treated, the route of administration, and the natureand severity of the condition to be treated. For example, according to aparticular embodiment, an initial dose of about 2 to 3 times themaintenance dose may suitably be administered about 4 to 12 hours beforetransplantation, followed by a daily dosage of 2 to 3 times themaintenance dose for one to two weeks, before gradually tapering down ata rate of about 5% a week to reach the maintenance dose.

The skilled person may determine those dosages that provide atherapeutic amount of an immunosuppressant agent at a level that istolerated. In a preferred embodiment, the method further comprisesadministering a single dose of antilymphocyte globulin, of about 1.6mg/20 g of body weight on the day of engraftment or transplantation. Inanother preferred embodiment, rapamycin may be applied at a dosage rangeof from about 0.05 to about 15 mg/kg/day, more preferably from about0.25 to about 5 mg/kg/day, and most preferably from about 0.5 to about1.5 mg/kg/day. Ideally, the administration of doses of one or more thanone immunosuppressant agent(s) can be curtailed after effectivetreatment with the DNA vaccine. Kits with same or different dosage formsmay be provided.

The following Examples provide further illustrations of the subjecttechnology and are not intended to limit the scope of the subjecttechnology.

EXAMPLIFICATION Example 1 Use of DNA Vaccine for the Treatment ofAlopecia Areata

A plasmid DNA construct encoding a pro-apoptotic protein is made. Thepro-apoptotic protein is selected from: Bax, Bak, Bim, Puma, Bad, Bik,Noxa, Bmf, Hrk, Bid, FAS, a caspase mutant, or a survivin mutant. Theplasmid is isolated from a bacterial strain that hypermethylates CpGdinucleotides and from a bacterial strain that does not hypermethylateCpG dinucleotides (i.e., hypomethylated). In the case of thehypermethylated DNA construct, the promoter controlling transcription ofthe gene/cDNA encoding the pro-apoptotic protein is not inactivated byCpG hypermethylation, e.g., the SV40 promoter. In the case of thenon-hypermethylated construct the promoter can be any inducible orconstitutive promoter, e.g., the CMV or LTR promoter.

Each of the plasmid DNA construct or different combinations of variousratios of the two constructs are then delivered to the skin to treatalopecia areata, e.g., using the C3H/HeJ mouse model. DNA is deliveredintradermally either in healthy skin, i.e., with hair, or directly intothe diseased area showing hair loss. DNA amounts can range from 1microgram to 200 microgram or more per treatment, and the treatment isdelivered weekly until hair growth is observed. Alternatively, the DNAcan be delivered transdermally after packaging into nano ormicroparticles that promote cell transfection.

In addition, the plasmid DNA construct can also encode an autoantigenpresent in hair follicles. In this case the DNA can be injected in skinwhich has no hair follicle or stem cells promoting the formation of hairfollicles.

Example 2 Preparation of E. Coli with a Constitutive Methylase Gene

Two different strains of the bacterium Escherichia coli, DH5-alpha andGM2929, were engineered to carry the sssI gene encoding the CpG DNAmethylase from Spiroplasma sp. strain MQ1 (M. SssI) (Renbaum et al.,1990 Nucleic Acids Res 18: 1145-1152) under the transcriptional controlof a constitutive promoter. For this purpose, two plasmid DNA constructspart of the CRIM vector system previously described by Haldimann andWanner were selected as molecular tools for chromosomal integration ofthe sssI gene (Haldimann and Wanner 2001 J. Bacteriology 183:6384-6393).

The two components of the CRIM system that were selected are the pAH162CRIM plasmid and the pAH123 CRIM helper plasmid. Plasmid pAH162 carriesa tetracycline resistance gene, a multiple cloning site, astrain-restricted origin of replication, and the DNA attachment sitefrom phage D80 (attPΦ80) which permits integration of the plasmid DNAinto the single attPΦ80 site found in an E. coli chromosome. PlasmidpAH123 is a helper plasmid DNA construct carrying an ampicillinresistance gene and the intΦ80 gene, which encodes the phage 080integrase that catalyzes recombination between attPΦ80 sites. Inaddition, expression of intΦ80 is induced after incubation of E. colicells at 37° C. which is a non-permissive temperature for plasmidpAH123, i.e., the plasmid DNA is lost. In contrast, growth at 30° C., ispermissive for the plasmid.

The initial plan for constructing the desired E. coli strains was tofollow the general protocol described by Haldimann and Wanner.Specifically, the strategy was to clone the sssI gene into plasmidpAH162 and then use the construct to transform E. coli DH5-alpha andGM2929 cells previously transformed with helper plasmid pAH123. Cellswould then be grown at 37° C. on medium containing the tetracyclineantibiotic. Growth at that temperature would result in the synthesis ofthe 080 integrase encoded by the helper plasmid, which would in turncause recombination between the pAH162-sssI plasmid and the E. colichromosome at the attPΦ80 site and chromosomal integration of theplasmid DNA. Because plasmid pAH162 carries tetracycline resistance andcannot replicate in strains DH5-alpha and GM2929, presence oftetracycline-resistant colonies would indicate chromosomal integrationof the plasmid DNA. In addition, helper plasmid pAH123 would be curedfrom the strains since it does not replicate at 37° C.

In order to clone the sssI gene into plasmid pAH162, a 280 bp BamHI-XbaIcontaining an undefined promoter sequence together the 5′ end of theopen-reading frame (ORF) coding for the SssI methylase was excised fromplasmid pAIT2 (New England Biolabs, Ipswich, Mass.) and cloned into theBamHI-XbaI restriction sites of plasmid pAH162, generating plasmidpAH162-1. Then, a 980 bp Xba-1-XbaI DNA fragment containing theremaining ORF for SssI was excised from pAIT2 and cloned into the XbaIsite of pAH162-1 in an effort to reconstitute the complete ORF codingfor SssI. However, no E. coli clones containing the reconstituted ORFwere obtained in spite of multiple attempts.

Because pAH162 and pAIT2 are high and low copy number plasmid DNA,respectively, it was hypothesized that the high copy number of the sssIgene carried by pAH162 was toxic to E. coli cells. Accordingly, a 1.3 kbPCR product containing the sssI gene and its undefined promoter sequencewas amplified from plasmid pAIT2 and cloned into the Seal site of thelow copy number plasmid pACYC184. An E. coli clone containing pACYC184carrying a 1.3 kb insert was obtained and restriction analysis indicatedthat the insert had the structural identity of the sssI gene. Moreover,restriction analysis also confirmed that the plasmid DNA construct hadincreased levels of methylated CpG dinucleotides. Specifically, pACYC184carrying the sssI gene (pACYC184-sssI) was isolated, digested with therestriction endonucleases HpaII and MspI, and analyzed using agarose gelelectrophoresis. The HpaII and MspI endonucleases do not and do,respectively, digest DNA when their respective target sequence isCpG-methylated. Results indicated that the HpaII enzyme did not digestpACYC184 when it carried the 1.3 kb insert, but that the MspI enzymedigested pACYC184 whether or not the plasmid DNA carried the 1.3 kbinsert. Therefore, results indicated that the 1.3 kb insert encoded afunctional SssI methylase and that the high number of pAH162 preventedcloning of the sssI gene in that plasmid. Together, these resultsindicated that plasmid pAH162-sssI could not be amplified in E. coli,thereby limiting its use for integration of the sssI gene into the E.coli chromosome. Accordingly, means of constructing and producingpAH162-sssI plasmid DNA without amplification in E. coli were developed.

The first strategy consisted of excising a 1.7 kb BamHI-BsaBI DNAfragment containing the sssI gene+promoter sequence from pACYC184-sssI,to isolate and ligate in vitro the DNA fragment into plasmid pAH162digested with BamHI-SmaI, and to use the ligation mixture to transformdirectly E. coli DH5-alpha cells already carrying the pAH123 helperplasmid for selection at 37° C. on tetracycline-containing agar plates.However, no colonies were obtained using this approach. Controlexperiments revealed that only one tetracycline-resistant colony couldbe obtained after electroporation of 250 nanograms of undigested vectorpAH162 alone, which indicated that an unrealistically large amount ofligation mixture would have to be used to obtain chromosomalintegration. Accordingly, a second strategy was taken with the goal ofobtaining large amounts of pAH162-sssI in vitro.

The second strategy consisted of cloning the 1.7 kb PCR fragmentcontaining the sssI gene+promoter DNA sequence into pAH162, to obtainlarge amounts of the desired circular plasmid pAH162-sssI plasmid DNAconstruct alone from the ligation mixture in order to transformDH5-alpha and select for chromosomal integration. It was important touse only the circular form of pAH162-sssI because linear DNA could notbe used for integration. Furthermore, it was also important to minimizecontamination from other plasmid DNA forms because of the inherent lowefficiency of chromosomal integration.

Accordingly, the following steps were devised and taken: The 1.7 kb PCRproduct containing the sssI gene+promoter DNA sequence was cloned intothe SmaI site of plasmid pAH162. As a result, two different orientationsof the 1.7 kb PCR insert were obtained. However, only one orientationcould be used to proceed with amplification because subsequentmanipulation of the amplified plasmid DNA would require digestion of theproduct with BamHI to obtain the linearized form of pAH162-sssI. Onlyone orientation of the PCR product could generate the linearized form ofpAH162-sssI. The other orientation would have generated a DNA fragmentcorresponding to the sssI gene together with a separate DNA fragmentcorresponding to plasmid pAH162.

To ensure that the desired orientation alone would serve as a substratefor amplification, the ligation mixture containing originally the 1.7 kbPCR product and SmaI-treated pAH162 was digested with BamHI. Twooligonucleotides were then used to amplify selectively the linearizedform of pAH162-sssI carrying the PCR insert in the desired orientation.The oligonucleotides hybridize to DNA sequences flanking one of the twoBamHI sites that are found in close proximity to each other in theligation product carrying the desired orientation of the PCR productinsert. Therefore, synthesis of the ligation product with desiredorientation is favored after PCR amplification. Following amplificationwith PCR, the 4.3 kb desired pAH162-sssI DNA product was digested withBamHI, fractionated using agarose gel electrophoresis, and directlyisolated from the gel. See the schematic diagram in FIG. 1 .

The isolated product was then used for its amplification in vitro inlarge amounts. Fifty nanograms of the isolated, BamHI-digestedpAH162-sssI DNA was re-ligated for the purpose of multi-primed rollingcircle amplification using the DNA polymerase from phage D29 accordingto a protocol derived from that described by Dean and co-workers (Deanet al., 2001 Genome Res 11: 1095). Specifically, 10 microliters ofdouble-distilled water containing 50 nanograms of religated pAH162-sssIplasmid DNA were mixed with 4.4 microliters of 10×029 DNA polymerasebuffer (New England Biolabs), 0.5 microliters of 10× bovine serumalbumin (0.1 milligram/milliliter final concentration), 4.4 microlitersof random hexamer oligonucleotides (New England Biolabs, 50 micromolarfinal concentration), 22 microliters of 2 millimolar of each of the 4dNTPs necessary for DNA synthesis, and double-distilled water for atotal volume of 42.9 microliters. The mixture was kept at 70° C. for 5minutes and then at 30° C. for 30 minutes to permit annealing of thehexamers to plasmid DNA strands. Following this incubation period, 1.1microliter of 029 DNA polymerase (New England Biolabs, 10,000units/milliliter) was added. The 44 microliters of prepared solution wasgently mixed and incubated for 3 hours at 30° C. The reaction volume wasthen doubled every 3 hours using a solution consisting of the sameoriginal components but without plasmid DNA template. After 12 hours ofincubation at 30° C., the reaction (352 microliters total volume) wasstopped after incubation at 70° C. for 10 minutes. DNA was precipitatedand total yield was estimated to be 140 micrograms using ultravioletspectrophotometry. Presence of the 1.7 kb DNA fragment containing thesssI gene was then confirmed using PCR analysis.

Nine micrograms of the amplification product was then digested withBamHI and religated. Agarose gel electrophoresis confirmed that thecorrect size of pAH162-sssI plasmid DNA product had been obtained afteramplification. Aliquots of one microgram of the re-ligated product werethen used for electroporation of E. coli DH5-alpha carrying the pAH123helper plasmid. Each microgram of the product yielded 2-4tetracycline-resistant colonies growing at 37° C., which was consistentwith our previous observation that 250 nanograms of vector pAH162yielded 1 tetracycline-resistant colony. In contrast, electroporation ofE. coli GM2929 cells with the same DNA product yielded ˜100 fold morecolonies. FIG. 1 shows the scheme used to successfully incorporate themethylase gene into E. coli under the control of a constitutivepromoter.

PCR analysis performed directly from bacterial cells confirmed thepresence of the 1.7 kb DNA fragment carrying the sssI gene in theobtained colonies. Furthermore, PCR analysis was also performed toconfirm integration and copy number of the 1.7 kb DNA fragment carryingthe sssI gene following a procedure described previously by Haldimannand Wanner (Haldimann and Wanner, 2001 J. Bacteriology 183: 6384).Specifically, four oligonucleotides (P1, P2, P3, and P4, with P1 and P4specific for the attPΦ80 integration site) were used together as primersin the PCR reaction. With these primers, a single DNA product indicatesno integration, two DNA products indicate single-copy integration, andthree DNA products indicate multiple integrations. Agarose gelelectrophoresis of the PCR reactions performed with 16 DH5-alpha clonesindicated that 14 clones contained a single integrated copy, one clonecontained multiple copies, and one clone contained no integrated copy ofthe desired insert. A similar analysis of nine GM2929 clones indicatedthat all clones contained a single integrated copy.

Levels of CpG-methylation of plasmid DNA isolated from E. coli clonescarrying a chromosomal copy of the sssI gene were then determined TwoDH-5a and two GM2929 clones carrying a single sssI gene insert wereselected and transformed with a plasmid DNA construct. In addition, thesame plasmid DNA construct was used to transform DH-5a and GM2929 cellsthat did not contain the sssI gene as control. Plasmid DNA was isolatedand sent to EpigenDX (Worcester, Mass.) for pyrosequencing anddetermination of levels of CpG methylation of 11 CpG dinucleotidepositions within the plasmid DNA. Results indicated mean CpG-methylationlevels of control plasmid DNA from DH5-alpha cells not expressing thesssI gene were ˜14-18% and that CpG-methylation levels from GM2929 cellsnot expressing the sssI gene were undetectable (See Table 1 below). Incontrast, mean CpG-methylation levels from DH5-alpha and GM2929 cellscarrying a single copy of the sssI gene were ˜47-51% and ˜49%,respectively. Together, these data show that E. coli strains carrying achromosomal copy of the sssI gene synthesize plasmid DNA with increasedlevels of methylated CpG dinucleotides.

TABLE 1 Sample Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Well ID #1 #2#3 #4 #5 #6 #7 #8 #9 #10 #11 Mean Stdev Min Max Note A1 DH 0.0 0.0 33.635.6 31.2 27.3 13.0 0.0 0.0 38.0 14.9 17.6 15.9 0.0 38.0 Low B1 DH 9.216.8 16.7 23.0 17.9 18.6 8.0 9.0 11.4 11.0 10.9 13.9 4.9 8.0 23.0 signalC1 GM — — — — — — — — — — — (—)No D1 3D 50.8 54.3 62.8 69.3 53.6 47.224.6 46.7 59.0 47.9 46.9 51.2 11.5 24.6 69.3 signal E1 16D 46.6 45.766.3 66.9 54.2 46.4 25.0 30.3 65.4 54.1 32.8 47.0 13.9 26.0 66.9 Low F116G 48.2 50.1 61.4 66.2 50.0 44.1 22.1 44.7 60.0 45.4 45.6 48.9 11.722.1 66.2 signal G1 17G 50.0 52.3 62.4 65.2 51.6 45.2 24.1 45.4 57.145.6 45.5 49.6 10.9 24.1 65.2

Table 1 shows the percentage of methylated CpG dinucleotides at 11positions and overall within a selected sequence of a plasmid DNA thatwas used to transform E. coli strains DH5-alpha (A1 and B1) and GM2929(C1) as controls, and DH5-alpha and GM2929 derivative strains carrying asingle chromosomal copy of the sssI gene, i.e., D1, E1 and F1, G1,respectively.

Example 3 Demonstration of Efficacy for Diabetes In Vivo

This Example and Example 6 show that partial hypermethylation of CpGdinucleotides carried by a plasmid DNA candidate product for therapy ofT1D is unexpectedly superior to full hypermethylation of the sameproduct for treatment of diabetes.

The DNA vaccines used in this Example and Example 6 composed of 2plasmid DNA constructs. One construct encodes the pro-apoptotic proteinBAX. The other construct encodes a secreted form of the pancreaticglutamic acid decarboxylase (sGAD), which is a T1D target autoantigen inboth NOD mice and humans. The cDNAs coding for sGAD and BAX are bothunder transcritptional control of the SV40 promoter, which is stillfunctional after CpG hypermethylation. Together, the 2 plasmid DNAconstructs constitute a single product candidate for treatment of T1D.

The product candidate encodes 3 immune components that modulate pathwaysnormally involved in maintaining immune tolerance and homeostasis inboth mice and humans. The first encoded component is the BAX proteinwhich induces formation of apoptotic cells in situ and which is namedADI for Apoptotic DNA Immunotherapy. ADI for induction of immunetolerance initiates the tolerogenic signal normally provided by theconstant flux of apoptotic cells processed daily by dendritic cells(DCs) which maintain immune tolerance to peripheral tissues in mammals.

The second encoded component is hypermethylation of CpG dinucleotides inplasmid DNA which modulates the innate immune response and results inincreased recruitment of plasmacytoid DCs (see below). Plasmid DNAhypermethylation is achieved using a proprietary strain of Escherichiacoli DH5-alpha that carries the SssI methylase gene in its chromosome.

The third encoded component is sGAD autoantigen which modulates theadaptive immune response through induction of Tregs when combined withADI. Both DCs and Tregs are essential leukocyte populations that controlinflammation and establish as well as maintain immune tolerance to selfantigens.

We previously showed that the 3 components are required in a synergisticmanner to treat spontaneously hyperglycemic NOD mice successfully. Newlyhyperglycemic mice, i.e., with different ages, received a weeklyintradermal (i.d.) injections of 40 micrograms hypermethylated plasmidDNA coding for sGAD (msGAD) with 10 micrograms hypomethylated plasmidDNA coding for BAX (BAX) over 8 weeks. Results indicated that 80% ofmice were diabetes-free at 40 weeks of age. Because of the beneficialeffect of hypermethylation of the plasmid DNA construct coding for sGADon treatment, we reasoned that hypermethylation of both plasmid DNAconstructs would lead to improved therapeutic efficacy. The hypothesiswas tested using treatment of 16-week-old NOD mice. Treating mice atthis age is stringent because DNA is delivered in animals with differentlevels of disease and beta-cell mass.

For 8 weeks, 16-week-old female NOD mice received a weekly intradermal(i.d.) injection of: 50 micrograms of DNA vaccines consisting of 40micrograms methylated plasmid DNA coding for secreted glutamic aciddecarboxylase (mSGAD) and 10 micrograms plasmid DNA coding for thepro-apoptotic protein BAX, where the DNA was eitherunmethylated/hypomethylated (BAX) or methylated/hypermethylated (mBAX).See Li et al. 2006 Vaccine 24: 5036-5046 and U.S. Pat. Pub. No.2008/0194510 (Escher et al.) for additional experimental details. It isexpected that about 10%-15% of CpG dinucleotides were methylated for the“BAX” group, and about 50% of CpG dinucleotides were methylated for the“mBAX” group. For convenience, the “msGAD+mBAX” group are sometimesreferred to as “fully” hypermethylated; and the “msGAD+BAX” group aresometimes referred to as “partially” hypermethylated.

In two groups, the plasmids were administered as 4:1 (4:1 ratio of theplasmid DNAs). 60 micrograms of DNA vaccines consisting of 40 microgramsmethylated plasmid DNA coding for secreted glutamic acid decarboxylase(mSGAD) and 20 micrograms plasmid DNA coding for pro-apoptotic proteinBAX, where the DNA was either unmethylated (BAX) or methylated (mBAX).In another two groups, the plasmids were administered as 4:2 (4:2 ratioof the plasmid DNAs). *P<0.05, Kaplan Meyer. FIG. 2 shows that thevaccines containing lower levels of methylation had higher therapeuticefficacy (previous work showed that unmethylated DNA vaccines had notherapeutic efficacy).

Particularly interesting is that injection of 40 micrograms msGAD with20 micrograms BAX (hypomethylated) significantly ameliorated disease.Unexpectedly, however, injection of 40 micrograms msGAD and 20micrograms mBAX (hypermethylated) did not. Therefore, hypermethylationof both plasmid DNA constructs actually caused a decrease in T1Dtreatment efficacy.

Furthermore, analysis of immune responses induced by injection of sGADwith BAX (hypomethylated), msGAD with BAX (hypomethylated), and msGADwith mBAX (hypermethylated) provided additional information on theeffects of DNA hypermethylation and supported the conclusion that a mixof hypomethylated and hypermethylated DNA is superior to hypomethylatedor hypermethylated DNA alone for induction of a tolerogenic immuneresponse.

Example 4 Demonstration of CD8+ Cell Penetration of Islet Cells In Vivo

Protocol: 10-week-old, 6 NOD/group, 3 NOD pooled, duplicated assay. Micewere treated as follows:

Group 1. Vector pMDV alone, 70 ug i.d. for 2 weeks, 3 times;

Group 2. SGAD+BAX=4:3, 70 ug i.d. for 2 weeks, 3 times; (no methylatedplasmid DNA);

Group 3. mSGAD+BAX=4:3, 70 ug i.d. for 2 weeks, 3 times; (40 ug plasmidDNA methylated); and

Group 4. mSGAD+mBAX, 70 ug i.d. for 2 weeks, 3 times; (70 ug plasmid DNAmethylated).

Islet Isolation & Culture

Isolation: Modified Pittsburg protocol, all hand-picked, no Ficoll,50-100 islets per NOD.

Culture: Islets were cultured in a 60 mm plate with T cell medium plusrhIL2 for 7 days (medium changed at day 4), islets were removed, andresuspended cells were collected by centrifugation.

FACS—Surface & Intracellular Staining: Collected cells were stained in10% FCS-PBS on ice for 30 min with anti-CD4-PE, anti-CD8-FITC, andanti-CD25-PECy5 Abs, and cells were washed. The Intracellular CellularStaining Kit (eBioscience, San Diego), was used: briefly, cells werefixed in Fixation/Permeabilization Solution for 30 min, washed, and thenanti-FoxP3-PECy5 or anti-IL17-PerCPCy5.5 plus Permeabilization Bufferwas applied for the other 30 min., washed, and cells were measured inFACS buffer by Flow Cytometer (Becton Dickinson, San Jose). PI stainingwas used to confirm unfixed cells that cell viabilities were higher than95%.

FIG. 3 shows the effects of DNA methylation levels on CD8+ T cellsinfiltration in pancreatic islets of immunized female NOD mice (T1Dmodel). Plasmid DNA was methylated using a DH5-alpha E. coli straincarrying a single copy of the sssI gene in its chromosome. FIG. 3further shows that increased DNA methylation causes decreasedinfiltration of islets by CD8+ T lymphocytes.

Example 5 Demonstration of Increased Regulatory T-Cell (Treg) PhenotypeIn Vivo

Protocol: 8-week-old, 6 NOD/group, duplicated assay. Mice were treatedas follows:

Group 1. pMDV NM+M=4:3, 70 ug i.d. for 2 weeks, 3 times;

Group 2. SGAD55+BAX=4:3, 70 ug i.d. for 2 weeks, 3 times;

Group 3. mSGAD55+BAX=4:3, 70 ug i.d. for 2 weeks, 3 times; and

Group 4. mSGAD55+mBAX=4:3, 70 ug i.d. for 2 weeks, 3 times.

LN & Spleen Isolation, Culture, & Separation

Axillary and pancreatic draining LN pooled, splenocytes were added tomake up to 4×107 total cells in 8 ml medium. For each set: cells wereloaded as 1 ml/well in a 24-well-plate for totally 8 wells, as 2 wellsfor No-Ag, 2 wells for Ins, and the last 2 wells for GAD Ag stimulation.Cells were cultured for 14 hrs with Ags plus CD154-PE Cocktail. Cellswere collected and Anti-PE-bead were used to separate CD154+ cells(Protocol of Miltenyi, Minn.). CD154+ were cultured w/CD3CD28Beads+rhIL2 for 3 days. Cells were stained with appropriateantibodies for flow cytometric analysis.

FIG. 4 shows the effects of DNA methylation levels on the percentage ofcells with Tregulatory cell phenotype in lymph nodes of immunized femaleNOD mice (T1D model). Plasmid DNA was methylated with a DH5-alpha E.coli strain carrying a single copy of the sssI gene in its chromosome.FIG. 4 further shows that increased DNA vaccine methylation causes anincreased percentage of cells with Treg phenotype.

Example 6 Demonstration of Efficacy for Allograph Survival In Vivo

Protocol: C57/B16 mice (8-week-old) recipients (n=10/group) receivedBALB/c full-thickness skingrafts on their back at day 0;

mice received Co60: Cobalt 3Gy at day 2, treated once;

Rapamycin (Wyeth, Madison, N.J.): 1 mg/kg BW daily until day 28; and

plasmid DNA 50 ug i.d., 1 cm from graft at days 0, 3, 7, and thenweekly.

Skin grafting was done as described in Li et al. 2010 Vaccine 28:1897-1904. See, Li et al. 2010 and U.S. Pat. Pub. No. 2009/0191218(Escher et al.) for additional experimental details.

FIG. 5 and FIG. 6 show the effect of CpG methylation of plasmid DNA onskin allograft survival. Plasmid DNA was methylated with a DH5-alpha E.coli strain carrying a single copy of the sssI gene in its chromosome.*, P<0.05, Kaplan Meyer.

Example 7 Modulating Plasmid DNA CpG-Methylation to Improve TolerogenicVaccination

The experimental setup is the same as Example 2. FIG. 7 shows thatinjection of fully hypermethylated (msGAD+mBAX) or partiallyhypermethylated DNA (msGAD+BAX) coding for sGAD and BAX promotedincreased recruitment of DCs, and of plasmacytoid DCs (pDCs) inparticular. Moreover, data indicate that the partially hypermethylatedDNA recruited highest numbers of pDCS. The results are significantbecause pDCs are associated with induction of tolerance and ameliorationof diabetes in NOD mice and humans.

FIG. 8 shows that DNA hypermethylation causes a shift in regulatory Tlymphocyte activity and that methylation levels modulate Treg function.Plasmid DNA hypermethylation has a significant impact on Tregs. Thefigure shows the effects of the same constructs described in FIG. 7 ,but this time on Treg activity in vivo after adoptive transfer of cellsfrom spleen of treated NOD mice mixed with diabetogenic T cells andinjected intravenously into NOD-scid mice which normally do not developdiabetes. Delayed diabetes in NOD-scid mice indicates suppressiveactivity of T cells from treated NOD mice acting on transferreddiabetogenic T cells. Results indicated that the non-hypermethylatedvaccine could induce CD4+CD25+ Tregs, although it does not amelioratedisease. Induction of Tregs in NOD mice without diabetes ameliorationhas been reported previously and suggests that Tregs may not be insufficient numbers, specificity, and overall activity for the treatmentto be effective in the vaccinated animal. Partial methylation of the DNA(msGAD+BAX or SKRS95) caused a shift in Treg population from CD4+CD25+to CD4+CD25−. These splenic CD4+CD25− cells may represent a populationof adaptive Tregs because newly activated (CD154+) GAD-specificCD4+CD25+FOXP3+ cells induced by GAD autoantigen were obtained fromcultured LNs of NOD mice receiving SKRS95. The fact that Tregstransferred from SKRS95-treated NOD mice did not show increasedamelioration of diabetes in NOD-scid compared to Tregs transferred fromNODs receiving the non-hypermethylated DNA suggests that SKRS95 inducesregulatory cell populations other than CD4+CD25−/CD25+ lymphocytes. Withregard to the fully hypermethylated msGAD+mBAX DNA, it did not inducedetectable splenic Treg activity in NOD-scid mice. Nonetheless, newlyactivated CD4+CD25+FOXP3+ cells could still be detected in culturedlymph nodes indicating that a certain level of Treg activity was inducedafter full DNA hypermethylation. Accordingly, partial methylation of theDNA (msGAD+BAX) caused a shift in Treg population from CD4+CD25+ toCD4+CD25−. The fully hypermethylated msGAD+mBAX DNA did not inducedetectable splenic Treg activity in vivo.

FIG. 9 shows flow cytometric analysis of cells from cultured pancreaticislets of mice receiving the different plasmid DNA constructs. The dataindicate that partially hypermethylated DNA was the only of the 4 testedDNAs (including control) that could cause a decrease in both CD4+IL17+and CD8+IL17+ cells which are also known as, respectively, Th17 and Tc17cells. An increasing body of evidence implicates Th17 lymphocytes in thepathogenesis of T1D in NOD mice, and downregulation of these cells isassociated with disease amelioration. As for Tc17 cells, they are asubgroup of CD8+ effector cells thought to have a pathogenic role inhuman autoimmune diseases like psoriasis, systemic lupus erythematosus,and immune thrombocytopenia. Furthermore, Tc17 cells have been shown tobe diabetogenic in a RIP-mOVA mouse model of T1D. Therefore, our findingthat msGAD with BAX cause a significant reduction in percentage of bothTh17 and Tc17 in cultured islets is significant.

Together, FIGS. 2 and 7-9 support the conclusion that partial, but notcomplete, hypermethylation of plasmid DNA coding for sGAD and BAXpromotes tolerogenic immune responses and treats diabetes successfully.

As mentioned previously, unmethylated CpG dinucleotides bind to the TLR9receptor which then signals the presence of bacterial DNA to themammalian host and promotes an inflammatory response. Accordingly, weinvestigated the role played by TLR9 in immune responses induced bymsGAD+BAX plasmid DNA.

FIG. 10 shows that co-injecting a CpG oligonucleotide that binds to TLR9together with mSGAD+BAX DNA caused a decrease in the in vitrosuppressive activity of pDCs isolated from treated NOD mice. These datasuggest that further activation of TLR9 by the CpG oligonucleotide wasdetrimental to tolerogenic-like immune responses induced by thepartially methylated plasmid DNA. Nevertheless, our previous data alsoclearly indicate that a certain level of unmethylated CpGs is necessaryfor therapeutic efficacy.

The unexpected finding that a certain level of unmethylated CpGdinucleotides is required to induce tolerogenic immune responses andsuccessfully treat an inflammatory disorder like T1D has significantimplications for the bench-to-bedside translation of our technology,because overall levels of TLR9 expression are known to be significantlydifferent between mice and humans. Therefore, CpG-methylation levelswill likely have to be adjusted for humans and presumably other speciesto optimize efficacy of treatment for a given disease in a givenspecies. Furthermore, CpG-methylation levels may also have to beadjusted for a given individual within a species in the context of aspecific disease. For example, kidney tissues from humans with lupusnephritis show higher levels of TLR9 compared with healthy individuals.Modulating level of unmethylated CpG dinucleotides for improved efficacyof immunotherapy would represent a novel means of personalized medicinefor the treatment of inflammatory disorders.

REFERENCES

-   Klinman D M, Yamshchikov G, Ishigatsubo Y. Contribution of CpG    motifs to the immunogenicity of DNA vaccines. J Immunol. 1997 Apr.    15; 158(8):3635-9.-   Reyes-Sandoval A, Ertl H C. CpG methylation of a plasmid vector    results in extended transgene product expression by circumventing    induction of immune responses. Mol Ther. 2004 February; 9(2):249-61.-   Ferguson T A, Choi J, Green D R. Armed response: how dying cells    influence T-cell functions. Immunol Rev. 2011 May; 241(1):77-88.-   Li A, Ojogho O, Franco E, Baron P, Iwaki Y, Escher A. Pro-apoptotic    DNA vaccination ameliorates new onset of autoimmune diabetes in NOD    mice and induces foxp3+ regulatory T cells in vitro. Vaccine. 2006    Jun. 5; 24(23):5036-46.-   Li A, Chen J, Hattori M, Franco E, Zuppan C, Ojogho O, Iwaki Y,    Escher A. A therapeutic DNA vaccination strategy for autoimmunity    and transplantation. Vaccine. 2010 Feb. 23; 28(8): 1897-904.-   Saxena V, Ondr J K, Magnusen A F, Munn D H, Katz J D. The    countervailing actions of myeloid and plasmacytoid dendritic cells    control autoimmune diabetes in the nonobese diabetic mouse. J.    Immunol. 2007 Oct. 15; 179(8):5041-53.-   Nikolic T, Welzen-Coppens J M, Leenen P J, Drexhage H A, Versnel    M A. Plasmacytoid dendritic cells in autoimmune diabetes—potential    tools for immunotherapy. Immunobiology. 2009; 214(9-10):791-9.-   Every A L, Kramer D R, Mannering S I, Lew A M, Harrison L C.    Intranasal vaccination with proinsulin DNAinduces regulatory CD4+ T    cells that prevent experimental autoimmune diabetes. J Immunol. 2006    Apr. 15; 176(8):4608-15.-   Jain R, Tartar D M, Gregg R K, Divekar R D, Bell J J, Lee H H, Yu P,    Ellis J S, Hoeman C M, Franklin C L, Zaghouani H. Innocuous IFNgamma    induced by adjuvant-free antigen restores normoglycemia in NOD mice    through inhibition of IL-17 production. J Exp Med. 2008 Jan. 21;    205(1):207-18.-   Emamaullee J A, Davis J, Merani S, Toso C, Elliott J F, Thiesen A,    Shapiro A M. Inhibition of Th17 cells regulates autoimmune diabetes    in NOD mice. Diabetes. 2009 June; 58(6):1302-11.-   Bertin-Maghit S, Pang D, O'Sullivan B, Best S, Duggan E, Paul S,    Thomas H, Kay T W, Harrison L C, Steptoe R, Thomas R. Interleukin-1β    produced in response to islet autoantigen presentation    differentiates T-helper 17 cells at the expense of regulatory    T-cells: Implications for the timing of tolerizing immunotherapy.    Diabetes. 2011 January;60(1):248-57.-   Zhang J, Huang Z, Sun R, Tian Z, Wei H. IFN-γ induced by IL-12    administration prevents diabetes by inhibiting pathogenic IL-17    production in NOD mice. J Autoimmun. 2012 February; 38(1):20-8.-   Res P C, Piskin G, de Boer O J, van der Loos C M, Teeling P, Bos J    D, Teunissen M B. Overrepresentation of IL-17A and IL-22 producing    CD8 T cells in lesional skin suggests their involvement in the    pathogenesis of psoriasis. PLoS One. 2010 Nov. 24; 5(11):e14108.-   Henriques A, Inês L, Couto M, Pedreiro S, Santos C, Magalhães M,    Santos P, Velada I, Almeida A, Carvalheiro T, Laranjeira P, Morgado    J M, Pais M L, da Silva J A, Paiva A. Frequency and functional    activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus    Erythematosus. Cell Immunol. 2010; 264(1):97-103.-   Hu Y, Ma D X, Shan N N, Zhu Y Y, Liu X G, Zhang L, Yu S, Ji C Y,    Hou M. Increased number of Tc17 and correlation with Th17 cells in    patients with immune thrombocytopenia. PLoS One. 2011; 6(10):e26522.-   Ciric B, El-behi M, Cabrera R, Zhang G X, Rostami A. IL-23 drives    pathogenic IL-17-producing CD8+ T cells. J Immunol. 2009 May 1;    182(9):5296-305.-   Mestas J, Hughes C C. Of mice and not men: differences between mouse    and human immunology. J Immunol. 2004 Mar. 1; 172(5):2731-8.-   Campbell J D, Cho Y, Foster M L, Kanzler H, Kachura M A, Lum J A,    Ratcliffe M J, Sathe A, Leishman A J, Bahl A, McHale M, Coffman R L,    Hessel E M. CpG-containing immunostimulatory DNA sequences elicit    TNF-alpha-dependent toxicity in rodents but not in humans. J Clin    Invest. 2009 September; 119(9):2564-76.

It is to be understood that, while the subject technology has beendescribed in conjunction with the detailed description, thereof, theforegoing description is intended to illustrate and not limit the scopeof the subject technology. Other aspects, advantages, and modificationsof the subject technology are within the scope of the claims set forthbelow. The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications,patents, and sequences cited in this disclosure are incorporated byreference in their entirety. To the extent the material incorporated byreference contradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

Appendix: SEQUENCES 1. Nucleotide sequence encoding human BAXatggacgggt ccggggagca gcccagaggc ggggggccca ccagctctga gcagatcatg 60aagacagggg cccttttgct tcagggtttc atccaggatc gagcagggcg aatggggggg 120gaggcacccg agctggccct ggacccggtg cctcaggatg cgtccaccaa gaagctgagc 180gagtgtctca agcgcatcgg ggacgaactg gacagtaaca tggagctgca gaggatgatt 240gccgccgtgg acacagactc cccccgagag gtctttttcc gagtggcagc tgacatgttt 300tctgacggca acttcaactg gggccgggtt gtcgcccttt tctactttgc cagcaaactg 360gtgctcaagg ccctgtgcac caaggtgccg gaactgatca gaaccatcat gggctggaca 420ttggacttcc tccgggagcg gctgttgggc tggatccaag accagggtgg ttgggacggc 480ctcctctcct actttgggac gcccacgtgg cagaccgtga ccatctttgt ggcgggagtg 540ctcaccgcct cgctcaccat ctggaagaag atgggctga 5792. Nucleotide sequence encoding human glutamic acid decarboxylase (GAD):gagctccacc gcggtggcgg ccgctctaga ccaccatggc atctccgggc tctggctttt 60ggtctttcgg gtcggaagat ggctctgggg attccgagaa tcccggcaca gcgcgagcct 120ggtgccaagt ggctcagaag ttcacgggcg gcatcggaaa caaactgtgc gccctgctct 180acggagacgc cgagaagccg gcggagagcg gcgggagcca acccccgcgg gccgccgccc 240ggaaggccgc ctgcgcctgc gaccagaagc cctgcagctg ctccaaagtg gatgtcaact 300acgcgtttct ccatgcaaca gacctgctgc cggcgtgtga tggagaaagg cccactttgg 360cgtttctgca agatgttatg aacattttac ttcagtatgt ggtgaaaagt ttcgatagat 420caaccaaagt gattgatttc cattatccta atgagcttct ccaagaatat aattgggaat 480tggcagacca accacaaaat ttggaggaaa ttttgatgca ttgccaaaca actctaaaat 540atgcaattaa aacagggcat cctagatact tcaatcaact ttctactggt ttggatatgg 600ttggattagc agcagactgg ctgacatcaa cagcaaatac taacatgttc acctatgaaa 660ttgctccagt atttgtgctt ttggaatatg tcacactaaa gaaaatgaga gaaatcattg 720gctggccagg gggctctggc gatgggatat tttctcccgg tggcgccata tctaacatgt 780atgccatgat gatcgcacgc tttaagatgt tcccagaagt caaggagaaa ggaatggctg 840ctcttcccag gctcattgcc ttcacgtctg aacatagtca tttttctctc aagaagggag 900ctgcagcctt agggattgga agagacagcg tgattctgat taaatgtgat gagagaggga 960aaatgattcc atctgatctt gaaagaagga ttcttgaagc caaacagaaa gggtttgttc 1020ctttcctcgt gagtgccaca gctggaacca ccgtgtacgg agcatttgac cccctcttag 1080ctgtcgctga catttgcaaa aagtataaga tctggatgca tgtggatgca gcttggggtg 1140ggggattact gatgtcccga aaacacaagt ggaaactgag tggcgtggag agggccaact 1200ctgtgacgtg gaatccacac aagatgatgg gagtcccttt gcagtggtct gctctcctgg 1260ttagagaaga gggattgatg cagaattgca accaaatgca tgcctcctac ctctttcagc 1320aagataaaca ttatgacctg tcctatgaca ctggagacaa ggccttacag tgcggacgcc 1380acgttgatgt ttttaaacta tggctgatgt ggagggcaaa ggggactacc gggtttgaag 1440cgcatgttga taaatgtttg gagttggcag agtatttata caacatcata aaaaaccgag 15003. Nucleotide sequence encoding a secreted form of human GAD (sGAD)atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60gcacctactt acgcgtttct ccatgcaaca gacctgctgc cggcgtgtga tggagaaagg 120cccactttgg cgtttctgca agatgttatg aacattttac ttcagtatgt ggtgaaaagt 180ttcgatagat caaccaaagt gattgatttc cattatccta atgagcttct ccaagaatat 240aattgggaat tggcagacca accacaaaat ttggaggaaa ttttgatgca ttgccaaaca 300actctaaaat atgcaattaa aacagggcat cctagatact tcaatcaact ttctactggt 360ttggatatgg ttggattagc agcagactgg ctgacatcaa cagcaaatac taacatgttc 420acctatgaaa ttgctccagt atttgtgctt ttggaatatg tcacactaaa gaaaatgaga 480gaaatcattg gctggccagg gggctctggc gatgggatat tttctcccgg tggcgccata 540tctaacatgt atgccatgat gatcgcacgc tttaagatgt tcccagaagt caaggagaaa 600ggaatggctg ctcttcccag gctcattgcc ttcacgtctg aacatagtca tttttctctc 660aagaagggag ctgcagcctt agggattgga agagacagcg tgattctgat taaatgtgat 720gagagaggga aaatgattcc atctgatctt gaaagaagga ttcttgaagc caaacagaaa 780gggtttgttc ctttcctcgt gagtgccaca gctggaacca ccgtgtacgg agcatttgac 840cccctcttag ctgtcgctga catttgcaaa aagtataaga tctggatgca tgtggatgca 900gcttggggtg ggggattact gatgtcccga aaacacaagt ggaaactgag tggcgtggag 960agggccaact ctgtgacgtg gaatccacac aagatgatgg gagtcccttt gcagtggtct 1020gctctcctgg ttagagaaga gggattgatg cagaattgca accaaatgca tgcctcctac 1080ctctttcagc aagataaaca ttatgacctg tcctatgaca ctggagacaa ggccttacag 1140tgcggacgcc acgttgatgt ttttaaacta tggctgatgt ggagggcaaa ggggactacc 1200gggtttgaag cgcatgttga taaatgtttg gagttggcag agtatttata caacatcata 1260aaaaaccgag aaggatatga gatggtgttt gatgggaagc ctgaggacac aaatgtctgc 1320ttctggtaca ttcctccaag cttgcgtact ctggaagaca atgaagagag aatgagtcgc 1380ctctcgaagg tggctccagt gattaaagcc agaatgatgg agtatggaac cacaatggtc 1440agctaccaac ccttgggaga caaggtcaat ttcttccgca tggtcatctc aaacccagcg 1500gcaactcacc aagacattga cttcctgatt gaagaaatag aacgccttgg acaagattta 15604. Nucleotide sequence of a constitutive promoter used in the Examples:5′GGATCCGCGTAATCATCGGCTCGTATAATGTGTGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACC 3′

What is claimed is:
 1. A method of stimulating hair growth, the methodcomprising administering to a human subject suffering hair loss from anautoimmune disorder an effective amount of a composition comprising apolynucleotide that encodes a Bax protein or a Bax pro-apoptoticfunctional fragment thereof, wherein the polynucleotide is: (a) operablylinked to an expression control element, and (b) methylated at CpGdinucleotides at a level that is about 2-fold to about 4-fold higher ascompared to the average methylation level of CpG dinucleotides in a wildtype Escherichia coli (E. coli) genome; and wherein the polynucleotideis administered intradermally directly to the site of hair loss,directly adjacent to the site of hair loss, directly to a site wherehair is present, or combinations thereof.
 2. The method of claim 1,wherein the polynucleotide that encodes the Bax protein or the Baxpro-apoptotic functional fragment thereof is in a vector and ismethylated in a bacterium comprising an exogenous polynucleotideencoding a DNA methyltransferase that is controlled by a constitutivepromoter, wherein the exogenous polynucleotide encoding the DNAmethyltransferase that is controlled by the constitutive promoter isstably incorporated into the chromosome of the bacterium.
 3. The methodof claim 2, wherein the bacterium is Escherichia coli (E. coli).
 4. Themethod of claim 2, wherein the encoded DNA methyltransferase comprises aCpG methylase.
 5. The method of claim 1, wherein the polynucleotidecomprises the Bax-encoding polynucleotide sequence of SEQ ID NO: 1, orthe polynucleotide comprises the portion of the polynucleotide of SEQ IDNO: 1 that encodes the pro-apoptotic functional fragment of Bax.
 6. Themethod of claim 5, wherein the polynucleotide is formulated as acomposition comprising the polynucleotide that encodes the Bax proteinor the Bax pro-apoptotic functional fragment thereof, a secondpolynucleotide encoding an autoantigen present in skin or a hairfollicle and operably linked to an expression control element, and apharmaceutically acceptable carrier.
 7. The method of claim 1, whereinthe polynucleotide is formulated as a composition comprising thepolynucleotide that encodes the Bax protein or the Bax pro-apoptoticfunctional fragment thereof, a second polynucleotide encoding anautoantigen present in skin or a hair follicle and operably linked to anexpression control element, and a pharmaceutically acceptable carrier.8. The method of claim 1, wherein the polynucleotide is formulatedwithin a microparticle or a nanoparticle.
 9. The method of claim 1,wherein the polynucleotide is formulated within a microparticle or ananoparticle comprising the polynucleotide that encodes the Bax proteinor the Bax pro-apoptotic functional fragment thereof, and a secondpolynucleotide encoding an autoantigen present in skin or a hairfollicle, wherein the second polynucleotide encoding the autoantigen isoperably linked to an expression control element.
 10. The method ofclaim 1, wherein the autoimmune disease is a chronic autoimmune disease.11. A method of stimulating growth of a hair shaft from a hair follicle,the method comprising administering to a human subject suffering from anautoimmune disorder, an effective amount of a polynucleotide thatencodes a Bax protein or a Bax pro-apoptotic functional fragment thereofand the polynucleotide is operably linked to an expression controlelement, wherein about 30% to about 60% of the CpG dinucleotides of thepolynucleotide are methylated, and wherein the polynucleotide isadministered intradermally directly to the site of hair loss, directlyadjacent to the site of hair loss, directly to a site where hair ispresent, or combinations thereof.
 12. The method of claim 11, whereinthe polynucleotide comprises the Bax-encoding polynucleotide sequence ofSEQ ID NO: 1, or the polynucleotide comprises the portion of thepolynucleotide of SEQ ID NO: 1 that encodes the pro-apoptotic functionalfragment of Bax.
 13. The method of claim 12, wherein the polynucleotideis formulated as a composition comprising the polynucleotide thatencodes the Bax protein or the Bax pro-apoptotic functional fragmentthereof, a second polynucleotide encoding an autoantigen present in skinor a hair follicle and operably linked to an expression control element,and a pharmaceutically acceptable carrier.
 14. The method of claim 11,wherein the polynucleotide is formulated as a composition comprising thepolynucleotide that encodes the Bax protein or the Bax pro-apoptoticfunctional fragment thereof, a second polynucleotide encoding anautoantigen present in skin or a hair follicle and operably linked to anexpression control element, and a pharmaceutically acceptable carrier.15. The method of claim 11, wherein the polynucleotide is formulatedwithin a microparticle or a nanoparticle.
 16. The method of claim 11,wherein the polynucleotide is formulated within a microparticle or ananoparticle comprising the polynucleotide that encodes the Bax proteinor the Bax pro-apoptotic functional fragment thereof, and a secondpolynucleotide encoding an autoantigen present in skin or a hairfollicle, wherein the second polynucleotide encoding the autoantigen isoperably linked to an expression control element.
 17. The method ofclaim 11, wherein the autoimmune disease is a chronic autoimmunedisease.